Protein (poly)peptides libraries

ABSTRACT

The present invention relates to synthetic DNA sequences which encode one or more collections of homologous proteins/(poly)peptides, and methods for generating and applying libraries of these DNA sequences. In particular, the invention relates to the preparation of a library of human-derived antibody genes by the use of synthetic consensus sequences which cover the structural repertoire of antibodies encoded in the human genome. Furthermore, the invention relates to the use of a single consensus antibody gene as a universal framework for highly diverse antibody libraries.

FIELD OF THE INVENTION

The present invention relates to synthetic DNA sequences which encodeone or more collections of homologous proteins/(poly)peptides, andmethods for generating and applying libraries of these DNA sequences. Inparticular, the invention relates to the preparation of a library ofhuman-derived antibody genes by the use of synthetic consensus sequenceswhich cover the structural repertoire of antibodies encoded in the humangenome. Furthermore, the invention relates to the use of a singleconsensus antibody gene as a universal framework for highly diverseantibody libraries.

BACKGROUND TO THE INVENTION

All current recombinant methods which use libraries ofproteins/(poly)peptides, e.g. antibodies, to screen for members withdesired properties, e.g. binding a given ligand, do not provide thepossibility to improve the desired properties of the members in an easyand rapid manner. Usually a library is created either by inserting arandom oligonucleotide sequence into one or more DNA sequences clonedfrom an organism, or a family of DNA sequences is cloned and used as thelibrary. The library is then screened, e.g. using phage display, formembers which show the desired property. The sequences of one or more ofthese resulting molecules are then determined. There is no generalprocedure available to improve these molecules further on.

Winter (EP 0 368 684 B1) has provided a method for amplifying (by PCR),cloning, and expressing antibody variable region genes. Starting withthese genes he was able to create libraries of functional antibodyfragments by randomizing the CDR3 of the heavy and/or the light chain.This process is functionally equivalent to the natural process of VJ andVDJ recombination which occurs during the development of B-cells in theimmune system.

However the Winter invention does not provide a method for optimizingthe binding affinities of antibody fragments further on, a process whichwould be functionally equivalent to the naturally occurring phenomenonof “affinity maturation”, which is provided by the present invention.Furthermore, the Winter invention does not provide for artificialvariable region genes, which represent a whole family of structurallysimilar natural genes, and which can be assembled from synthetic DNAoligonucleotides. Additionally, Winter does not enable the combinatorialassembly of portions of antibody variable regions, a feature which isprovided by the present invention. Furthermore, this approach has thedisadvantage that the genes of all antibodies obtained in the screeningprocedure have to be completely sequenced, since, except for the PCRpriming regions, no additional sequence information about the librarymembers is available. This is time and labor intensive and potentiallyleads to sequencing errors.

The teaching of Winter as well as other approaches have tried to createlarge antibody libraries having high diversity in the complementaritydetermining regions (CDRs) as well as in the frameworks to be able tofind antibodies against as many different antigens as possible. It hasbeen suggested that a single universal framework may be useful to buildantibody libraries, but no approach has yet been successful.

Another problem lies in the production of reagents derived fromantibodies. Small antibody fragments show exciting promise for use astherapeutic agents, diagnostic reagents, and for biochemical research.Thus, they are needed in large amounts, and the expression of antibodyfragments, e.g. Fv, single-chain Fv (scFv), or Fab in the periplasm ofE. coli (Skerra & Plückthun, 1988; Better et al., 1988) is now usedroutinely in many laboratories. Expression yields vary widely, however.While some fragments yield up to several mg of functional, solubleprotein per liter and OD of culture broth in shake flask culture (Carteret al., 1992, Plückthun et al. 1996), other fragments may almostexclusively lead to insoluble material, often found in so-calledinclusion bodies. Functional protein may be obtained from the latter inmodest yields by a laborious and time-consuming refolding process. Thefactors influencing antibody expression levels are still only poorlyunderstood. Folding efficiency and stability of the antibody fragments,protease lability and toxicity of the expressed proteins to the hostcells often severely limit actual production levels, and severalattempts have been tried to increase expression yields. For example,Knappik & Plückthun (1995) could show that expression yield depends onthe antibody sequence. They identified key residues in the antibodyframework which influence expression yields dramatically. Similarly,Ullrich et al. (1995) fount that point mutations in the CDRs canincrease the yields in periplasmic antibody fragment expression.Nevertheless, these strategies are only applicable to a few antibodies.Since the Winter invention uses existing repertoires of antibodies, noinfluence on expressibility of the genes is possible.

Furthermore, the findings of Knappik & Plückthun ard Ullrich demonstratethat the knowledge about antibodies, especially about folding andexpression is still increasing. The Winter invention does not allow toincorporate such improvements into the library design.

The expressibility of the genes is important for the library quality aswell, since the screening procedure relies in most cases on the displayof the gene product on a phage surface, and efficient display relies onat least moderate expression of the gene.

These disadvantages of the existing methodologies are overcome by thepresent invention, which is applicable for all collections of homologousproteins. It has the following novel and useful features illustrated inthe following by antibodies as an example:

Artificial antibodies and fragments thereof can be constructed based onknown antibody sequences, which reflect the structural properties of awhole group of homologous antibody genes. Therefore it is possible toreduce the number of different genes without any loss in the structuralrepertoire. This approach leads to a limited set of artificial genes,which can be synthesized de novo, thereby allowing introduction ofcleavage sites and removing unwanted cleavages sites. Furthermore, thisapproach enables (i), adapting the codon usage of the genes to that ofhighly expressed genes in any desired host cell and (ii), analyzing allpossible pairs of antibody light (L) and heavy (H) chains in terms ofinteraction preference, antigen preference or recombinant expressiontiter, which is virtually impossible using the complete collection ofantibody genes of an organism and all combinations thereof.

The use of a limited set of completely synthetic genes makes it possibleto create cleavage sites at the boundaries of encoded structuralsub-elements. Therefore, each gene is built up from modules whichrepresent structural sub-elements on the protein/(poly)peptide level. Inthe case of antibodies, the modules consist of “framework” and “CDR”modules. By creating separate framework and CDR modules, differentcombinatorial assembly possibilities are enabled. Moreover, if two ormore artificial genes carry identical pairs of cleavage sites at theboundaries of each of the genetic sub-elements, pre-built libraries ofsub-elements can be inserted in these genes simultaneously, without anyadditional information related to any particular gene sequence. Thisstrategy enables rapid optimization of, for example, antibody affinity,since DNA cassettes encoding libraries of genetic sub-elements can be(i), pre-built, stored and reused and (ii), inserted in any of thesesequences at the right position without knowing the actual sequence orhaving to determine the sequence of the individual library member.

Additionally, new information about amino acid residues important forbinding, stability, or solubility and expression could be integratedinto the library design by replacing existing modules with modulesmodified according to the new observations.

The limited number of consensus sequences used for creating the libraryallows to speed up the identification of binding antibodies afterscreening. After having identified the underlying consensus genesequence, which could be done by sequencing or by using fingerprintrestriction sites, just those part(s) comprising the random sequence(s)have to be determined. This reduces the probability of sequencing errorsand of false-positive results.

The above mentioned cleavage sites can be used only if they are uniquein the vector system where the artificial genes have been inserted. As aresult, the vector has to be modified to contain none of these cleavagesites. The construction of a vector consisting of basic elements likeresistance gene and origin of replication, where cleavage sites havebeen removed, is of general interest for many cloning attempts.Additionally, these vector(s) could be part of a kit comprising theabove mentioned artificial genes and pre-built libraries.

The collection of artificial genes can be used for a rapid humanizationprocedure of non-human antibodies, preferably of rodent antibodies.First, the amino acid sequence of the non-human, preferably rodentantibody is compared with the amino acid sequences encoded by thecollection of artificial genes to determine the most homologous lightand heavy framework regions. These genes are then used for insertion ofthe genetic sub-elements encoding the CDRs of the non-human, preferablyrodent antibody.

Surprisingly, it has been found that with a combination of only oneconsensus sequence for each of the light and heavy chains of a scFvfragment an antibody repertoire could be created yielding antibodiesagainst virtually every antigen. Therefore, one aspect of the presentinvention is the use of a single consensus sequence as a universalframework for the creation of useful (poly)peptide libraries andantibody consensus sequences useful therefor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention enables the creation of useful libraries of(poly)peptides. In a first embodiment, the invention provides for amethod of setting up nucleic acid sequences suitable for the creation ofsaid libraries. In a first step, a collection of at least threehomologous proteins is identified and then analyzed. Therefore, adatabase of the protein sequences is established where the proteinsequences are aligned to each other. The database is used to definesubgroups of protein sequences which show a high degree of similarity inboth the sequence and, if information is available, in the structuralarrangement. For each of the subgroups a (poly)peptide sequencecomprising at least one consensus sequence is deduced which representsthe members of this subgroup; the complete collection of (poly)peptidesequences represent therefore the complete structural repertoire of thecollection of homologous proteins. These artificial (poly)peptidesequences are then analyzed, if possible, according to their structuralproperties to identify unfavorable interactions between amino acidswithin said (poly)peptide sequences or between said or other(poly)peptide sequences, for example, in multimeric proteins. Suchinteractions are then removed by changing the consensus sequenceaccordingly. The (poly)peptide sequences are then analyzed to identifysub-elements such as domains, loops, helices or CDRs. The amino acidsequence is backtranslated into a corresponding coding nucleic acidsequence which is adapted to the codon usage of the host planned forexpressing said nucleic acid sequences. A set of cleavage sites is setup in a way that each of the sub-sequences encoding the sub-elementsidentified as described above, is flanked by two sites which do notoccur a second time within the nucleic acid sequence. This can beachieved by either identifying a cleavage site already flanking asub-sequence of by changing one or more nucleotides to create thecleavage site, and by removing that site from the remaining part of thegene. The cleavage sites should be common to all correspondingsub-elements or sub-sequences, thus creating a fully modular arrangementof the sub-sequences in the nucleic acid sequence and of thesub-elements in the corresponding (poly)peptide.

In a further embodiment, the invention provides for a method which setsup two or more sets of (poly)peptides, where for each set the method asdescribed above is performed, and where the cleavage sites are not onlyunique within each set but also between any two sets. This method can beapplied for the creation of (poly)peptide libraries comprising forexample two α-helical domains from two different proteins, where saidlibrary is screened for novel hetero-association domains.

In yet a further embodiment, at least two of the sets as describedabove, are derived from the same collection of proteins or at least apart of it. This describes libraries comprising for example, but notlimited to, two domains from antibodies such as VH and VL, or twoextracellular loops of transmembrane receptors.

In another embodiment, the nucleic acid sequences set up as describedabove, are synthesized. This can be achieved by any one of severalmethods well known to the practitioner skilled in the art, for example,by total gene synthesis or by PCR-based approaches.

In one embodiment, the nucleic acid sequences are cloned into a vector.The vector could be a sequencing vector, an expression vector or adisplay (e.g. phage display) vector, which are well known to thoseskilled in the art. Any vector could comprise one nucleic acid sequence,or two or more nucleic sequences, either in different or the sameoperon. In the last case, they could either be cloned separately or ascontiguous sequences.

In one embodiment, the removal of unfavorable interactions as describedabove, leads to enhanced expression of the modified (poly)peptides.

In a preferred embodiment, one or more sub-sequences of the nucleic acidsequences are replaced by different sequences. This can be achieved byexcising the sub-sequences using the conditions suitable for cleavingthe cleavage sites adjacent to or at the end of the sub-sequence, forexample, by using a restriction enzyme at the corresponding restrictionsite under the conditions well known to those skilled in the art, andreplacing the sub-sequence by a different sequence compatible with thecleaved nucleic acid sequence. In a further preferred embodiment, thedifferent sequences replacing the initial sub-sequence(s) are genomic orrearranged genomic sequences, for example in grafting CDRs fromnon-human antibodies onto consensus antibody sequences for rapidhumanization of non-human antibodies. In the most preferred embodiment,the different sequences are random sequences, thus replacing thesub-sequence by a collection of sequences to introduce variability andto create a library. The random sequences can be assembled in variousways, for example by using a mixture of mononucleotides or preferably amixture of trinucleotides (Virnekäs et al., 1994) during automatedoligonucleotide synthesis, by error-prone PCR or by other methods wellknown to the practitioner in the art. The random sequences may becompletely randomized or biased towards or against certain codonsaccording to the amino acid distribution at certain positions in knownprotein sequences. Additionally, the collection of random sub-sequencesmay comprise different numbers of codons, giving rise to a collection ofsub-elements having different lengths.

In another embodiment, the invention provides for the expression of thenucleic acid sequences from a suitable vector and under suitableconditions well known to those skilled in the art.

In a further preferred embodiment, the (poly)peptides expressed fromsaid nucleic acid sequences are screened and, optionally, optimized.Screening may be performed by using one of the methods well known to thepractitioner in the art, such as phage-display, selectively infectivephage, polysome technology to screen for binding, assay systems forenzymatic activity or protein stability. (Poly)peptides having thedesired property can be identified by sequencing of the correspondingnucleic acid sequence or by amino acid sequencing or mass spectrometry.In the case of subsequent optimization, the nucleic acid sequencesencoding the initially selected (poly)peptides can optionally be usedwithout sequencing. Optimization is performed by repeating thereplacement of sub-sequences by different sequences, preferably byrandom sequences, and the screening step one or more times.

The desired property the (poly)peptides are screened for is preferably,but not exclusively, selected from the group of optimized affinity orspecificity for a target molecule, optimized enzymatic activity,optimized expression yields, optimized stability and optimizedsolubility.

In one embodiment, the cleavage sites flanking the sub-sequences aresites recognized and cleaved by restriction enzymes, with recognitionand cleavage sequences being either identical or different, therestricted sites either having blunt or sticky ends.

The length of the sub-elements is preferably, but not exclusivelyranging between 1 amino acid, such as one residue in the active site ofan enzyme or a structure-determining residue, and 150 amino acids, asfor whole protein domains. Most preferably, the length ranges between 3and 25 amino acids, such as most commonly found in CDR loops ofantibodies.

The nucleic acid sequences could be RNA or, preferably, DNA.

In one embodiment, the (poly)peptides have an amino acid patterncharacteristic of a particular species. This can for example be achievedby deducing the consensus sequences from a collection of homologousproteins of just one species, most preferably from a collection of humanproteins. Since the (poly)peptides comprising consensus sequences areartificial, they have to be compared to the protein sequence(s) havingthe closest similarity to ensure the presence of said characteristicamino acid pattern.

In one embodiment, the invention provides for the creation of librariesof (poly)peptides comprising at least part of members or derivatives ofthe immunoglobulin superfamily, preferably of member or derivatives ofthe immunoglobulins. Most preferably, the invention provides for thecreation of libraries of human antibodies, wherein said (poly)peptidesare or are derived from heavy or light chain variable regions whereinsaid structural sub-elements are framework regions (FR) 1, 2, 3, or 4 orcomplementary determining regions (CDR) 1, 2, or 3. In a first step, adatabase of published antibody sequences of human origin is establishedwhere the antibody sequences are aligned to each other. The database isused to define subgroups of antibody sequences which show a high degreeof similarity in both the sequence and the canonical fold of CDR loops(as determined by analysis of antibody structures). For each of thesubgroups a consensus sequence is deduced which represents the membersof this subgroup; the complete collection of consensus sequencesrepresent therefore the complete structural repertoire of humanantibodies.

These artificial genes are then constructed e.g. by total gene synthesisor by the use of synthetic genetic subunits. These genetic subunitscorrespond to structural sub-elements on the (poly)peptide level. On theDNA level, these genetic subunits are defined by cleavage sites at thestart and the end of each of the sub-elements, which are unique in thevector system. All genes which are members of the collection ofconsensus sequences are constructed such that they contain a similarpattern of corresponding genetic sub-sequences. Most preferably, said(poly)peptides are or are derived from the HuCAL consensus genes: Vκ1,Vκ2, Vκ3, Vκ4, Vλ1, Vλ2, Vλ3, VH1A, VH1B, VH2, VH3, VH4, VH5, VH6, Cκ,Cπ, CH1 or any combination of said HuCAL consensus genes.

This collection of DNA molecules can then be used to create libraries ofantibodies or antibody fragments, preferably Fv, disulphide-linked Fv,single-chain Fv (scFv), or Fab fragments, which may be used as sourcesof specificities against new target antigens. Moreover, the affinity ofthe antibodies can be optimized using pre-built library cassettes and ageneral procedure. The invention provides a method for identifying oneor more genes encoding one or more antibody fragments which binds to atarget, comprising the steps of expressing the antibody fragments, andthen screening them to isolate one or more antibody fragments which bindto a given target molecule. Preferably, an scFv fragment librarycomprising the combination of HuCAL VH3 and HuCAL Vλ2 consensus genesand at least a random sub-sequence encoding the heavy chain CDR3sub-element is screened for binding antibodies. If necessary, themodular design of the genes can then be used to excise from the genesencoding the antibody fragments one or more genetic sub-sequencesencoding structural sub-elements, and replacing them by one or moresecond sub-sequences encoding structural sub-elements. The expressionand screening steps can then be repeated until an antibody having thedesired affinity is generated.

Particularly preferred is a method in which one or more of the geneticsubunits (e.g. the CDRs) are replaced by a random collection ofsequences (the library) using the said cleavage sites. Since thesecleavage sites are (i) unique in the vector system and (ii) common toall consensus genes, the same (pre-built) library can be inserted intoall artificial antibody genes. The resulting library is then screenedagainst any chosen antigen. Binding antibodies are selected, collectedand used as starting material for the next library. Here, one or more ofthe remaining genetic subunits are randomized as described above.

A further embodiment of the present invention relates to fusion proteinsby providing for a DNA sequence which encodes both the (poly)peptide, asdescribed above, as well as an additional moiety. Particularly preferredare moieties which have a useful therapeutic function. For example, theadditional moiety may be a toxin molecule which is able to kill cells(Vitetta et al., 1993). There are numerous examples of such toxins, wellknown to the one skilled in the art, such as the bacterial toxinsPseudomonas exotoxin A, and diphtheria toxin, as well as the planttoxins ricin, abrin, modeccin, saporin, and gelonin. By fusing such atoxin for example to an antibody fragment, the toxin can be targeted to,for example, diseased cells, and thereby have a beneficial therapeuticeffect. Alternatively, the additional moiety may be a cytokine, such asIL-2 (Rosenberg & Lotze, 1986), which has a particular effect (in thiscase a T-cell proliferative effect) on a family of cells. In a furtherembodiment, the additional moiety may confer on its (poly)peptidepartner a means of detection and/or purification. For example, thefusion protein could comprise the modified antibody fragment and anenzyme commonly used for detection purposes, such as alkalinephosphatase (Blake et al., 1984). There are numerous other moietieswhich can be used as detection or purification tags, which are wellknown to the practitioner skilled in the art. Particularly preferred arepeptides comprising at least five histidine residues (Hochuli et al.,1988), which are able to bind to metal ions, and can therefore be usedfor the purification of the protein to which they are fused (Lindner etal., 1992). Also provided for by the invention are additional moietiessuch as the commonly used C-myc and FLAG tags (Hopp et al., 1988;Knappik & Plückthun, 1994).

By engineering one or more fused additional domains, antibody fragmentsor any other (poly)peptide can be assembled into larger molecules whichalso fall under the scope of the present invention. For example,mini-antibodies (Pack, 1994) are dimers comprising two antibodyfragments, each fused to a self-associating dimerization domain.Dimerization domains which are particularly preferred include thosederived from a leucine zipper (Pack & Plückthun, 1992) orhelix-turn-helix motif (Pack et al., 1993).

All of the above embodiments of the present invention can be effectedusing standard techniques of molecular biology known to anyone skilledin the art.

In a further embodiment, the random collection of sub-sequences (thelibrary) is inserted into a singular nucleic acid sequence encoding one(poly)peptide, thus creating a (poly)peptide library based on oneuniversal framework. Preferably a random collection of CDR sub-sequencesis inserted into a universal antibody framework, for example into theHuCAL H3κ2 single-chain Fv fragment described above.

In further embodiments, the invention provides for nucleic acidsequence(s), vector(s) containing the nucleic acid sequence(s), hostcell(s) containing the vector(s), and (poly)peptides, obtainableaccording to the methods described above.

In a further preferred embodiment, the invention provides for modularvector systems being compatible with the modular nucleic acid sequencesencoding the (poly)peptides. The modules of the vectors are flanked byrestriction sites unique within the vector system and essentially uniquewith respect to the restriction sites incorporated into the nucleic acidsequences encoding the (poly)peptides, except for example therestriction sites necessary for cloning the nucleic acid sequences intothe vector. The list of vector modules comprises origins ofsingle-stranded replication, origins of double-stranded replication forhigh- and low copy number plasmids, promotor/operator, repressor orterminator elements, resistance genes, potential recombination sites,gene III for display on filamentous phages, signal sequences,purification and detection tags, and sequences of additional moieties.

The vectors are preferably, but not exclusively, expression vectors orvectors suitable for expression and screening of libraries.

In another embodiment, the invention provides for a kit, comprising oneor more of the list of nucleic acid sequence(s), recombinant vector(s),(poly)peptide(s), and vector(s) according to the methods describedabove, and suitable host cell(s) for producing the (poly)peptide(s).

In a preferred embodiment, the invention provides for the creation oflibraries of human antibodies. In a first step, a database of publishedantibody sequences of human origin is established: The database is usedto define subgroups of antibody sequences which show a high degree ofsimilarity in both the sequence and the canonical fold (as determined byanalysis of antibody structures). For each of the subgroups a consensussequence is deduced which represents the members of this subgroup; thecomplete collection of consensus sequences represent therefore thecomplete structural repertoire of human antibodies.

These artificial genes are then constructed by the use of syntheticgenetic subunits. These genetic subunits correspond to structuralsub-elements on the protein level. On the DNA level, these geneticsubunits are defined by cleavage sites at the start and the end of eachof the subelements, which are unique in the vector system. All geneswhich are members of the collection of consensus sequences areconstructed such that they contain a similar pattern of said geneticsubunits.

This collection of DNA molecules can then be used to create libraries ofantibodies which may be used as sources of specificities against newtarget antigens. Moreover, the affinity of the antibodies can beoptimised using pre-built library cassettes and a general procedure. Theinvention provides a method for identifying one or more genes encodingone or more antibody fragments which binds to a target, comprising thesteps of expressing the antibody fragments, and then screening them toisolate one or more antibody fragments which bind to a given targetmolecule. If necessary, the modular design of the genes can then be usedto excise from the genes encoding the antibody fragments one or moregenetic sub-sequences encoding structural sub-elements, and replacingthem by one or more second sub-sequences encoding structuralsub-elements. The expression and screening steps can then be repeateduntil an antibody having the desired affinity is generated.

Particularly preferred is a method in which one or more or the geneticsubunits (e.g. the CDR's) are replaced by a random collection ofsequences (the library) using the said cleavage sites. Since thesecleavage sites are (i) unique in the vector system and (ii) common toall consensus genes, the same (pre-built) library can be inserted intoall artificial antibody genes. The resulting library is then screenedagainst any chosen antigen. Binding antibodies are eluted, collected andused as starting material for the next library. Here, one or more of theremaining genetic subunits are randomised as described above.

Definitions

Protein:

The term protein comprises monomeric polypeptide chains as well as homo-or heteromultimeric complexes of two or more polypeptide chainsconnected either by covalent interactions (such as disulphide bonds) orby non-covalent interactions (such as hydrophobic or electrostaticinteractions).

Analysis of Homologous Proteins:

The amino acid sequences of three or more proteins are aligned to eachother (allowing for introduction of gaps) in a way which maximizes thecorrespondence between identical or similar amino acid residues at allpositions. These aligned sequences are termed homologous if thepercentage of the sum of identical and/or similar residues exceeds adefined threshold. This threshold is commonly regarded by those skilledin the art as being exceeded when at least 15% of the amino acids in thealigned genes are identical, and at least 30% are similar. Examples forfamilies of homologous proteins are: immunoglobulin superfamily,scavenger receptor superfamily, fibronectin superfamilies (e.g. type IIand III), complement control protein superfamily, cytokine receptorsuperfamily, cystine knot proteins, tyrosine kinases, and numerous otherexamples well known to one of ordinary skill in the art.

Consensus Sequence:

Using a matrix of at least three aligned amino acid sequences, andallowing for gaps in the alignment, it is possible to determine the mostfrequent amino acid residue at each position. The consensus sequence isthat sequence which comprises the amino acids which are most frequentlyrepresented at each position. In the event that two or more amino acidsare equally represented at a single position, the consensus sequenceincludes both or all of those amino acids.

Removing Unfavorable Interactions:

The consensus sequence is per se in most cases artificial and has to beanalyzed in order to change amino acid residues which, for example,would prevent the resulting molecule to adapt a functional tertiarystructure or which would block the interaction with other (poly)peptidechains in multimeric complexes. This can be done either by (i) buildinga three-dimensional model of the consensus sequence using known relatedstructures as a template, and identifying amino acid residues within themodel which may interact unfavorably with each other, or (ii) analyzingthe matrix of aligned amino acid sequences in order to detectcombinations of amino acid residues within the sequences whichfrequently occur together in one sequence and are therefore likely tointeract with each other. These probable interaction-pairs are thentabulated and the consensus is compared with these “interaction maps”.Missing or wrong interactions in the consensus are repaired accordinglyby introducing appropriate changes in amino acids which minimizeunfavorable interactions.

Identification of Structural Sub-Elements:

Structural sub-elements are stretches of amino acid residues within aprotein/(poly)peptide which correspond to a defined structural orfunctional part of the molecule. These can be loops (e.g. CDR loops ofan antibody) or any other secondary or functional structure within theprotein/(poly)peptide (domains, α-helices, β-sheets, framework regionsof antibodies, etc.). A structural sub-element can be identified usingknown structures of similar or homologous (poly)peptides, or by usingthe above mentioned matrices of aligned amino acid sequences. Here thevariability at each position is the basis for determining stretches ofamino acid residues which belong to a structural sub-element (e.g.hypervariable regions of an antibody).

Sub-Sequence:

A sub-sequence is defined as a genetic module which is flanked by uniquecleavage sites and encodes at least one structural sub-element. It isnot necessarily identical to a structural sub-element.

Cleavage Site:

A short DNA sequence which is used as a specific target for a reagentwhich cleaves DNA in a sequence-specific manner (e.g. restrictionendonucleases).

Compatible Cleavage Sites:

Cleavage sites are compatible with each other, if they can beefficiently ligated without modification and, preferably, also withoutadding an adapter molecule.

Unique Cleavage Sites:

A cleavage site is defined as unique if it occurs only once in a vectorcontaining at least one of the genes of interest, or if a vectorcontaining at least one of the genes of interest could be treated in away that only one of the cleavage sites could be used by the cleavingagent.

Corresponding (Poly)Peptide Sequences:

Sequences deduced from the same part of one group of homologous proteinsare called corresponding (poly)peptide sequences.

Common Cleavage Sites:

A cleavage site in at least two corresponding sequences, which occurs atthe same functional position (i.e. which flanks a defined sub-sequence),which can be hydrolyzed by the same cleavage tool and which yieldsidentical compatible ends is termed a common cleavage site.

Excising Genetic Sub-Sequences:

A method which uses the unique cleavage sites and the correspondingcleavage reagents to cleave the target DNA at the specified positions inorder to isolate, remove or replace the genetic sub-sequence flanked bythese unique cleavage sites.

Exchanging Genetic Sub-Sequences:

A method by which an existing sub-sequence is removed using the flankingcleavage sites of this sub-sequence, and a new sub-sequence or acollection of sub-sequences, which contain ends compatible with thecleavage sites thus created, is inserted.

Expression of Genes:

The term expression refers to in vivo or in vitro processes, by whichthe information of a gene is transcribed into mRNA and then translatedinto a protein/(poly)peptide. Thus, the term expression refers to aprocess which occurs inside cells, by which the information of a gene istranscribed into mRNA and then into a protein. The term expression alsoincludes all events of post-translational modification and transport,which are necessary for the (poly)peptide to be functional.

Screening of Protein/(Poly)Peptide Libraries:

Any method which allows isolation of one or more proteins/(poly)peptideshaving a desired property from other proteins/(poly)peptides within alibrary.

Amino Acid Pattern Characteristic for a Species:

A (poly)peptide sequence is assumed to exhibit an amino acid patterncharacteristic for a species if it is deduced from a collection ofhomologous proteins from just this species.

Immunoglobulin Superfamily (IgSF):

The IgSF is a family of proteins comprising domains being characterizedby the immunoglobulin fold. The IgSF comprises for example T-cellreceptors and the immunoglobulins (antibodies).

Antibody Framework:

A framework of an antibody variable domain is defined by Kabat et al.(1991) as the part of the variable domain which serves as a scaffold forthe antigen binding loops of this variable domain.

Antibody CDR:

The CDRs (complementarity determining regions) of an antibody consist ofthe antigen binding loops, as defined by Kabat et al. (1991). Each ofthe two variable domains of an antibody Fv fragment contain three CDRs.

HuCAL:

Acronym for Human Combinatorial Antibody Library. Antibody Library basedon modular consensus genes according to the invention (see Example 1).

Antibody Fragment:

Any portion of an antibody which has a particular function, e.g. bindingof antigen. Usually, antibody fragments are smaller than wholeantibodies. Examples are Fv, disulphide-linked Fv, single-chain Fv(scFv), or Fab fragments. Additionally, antibody fragments are oftenengineered to include new functions or properties.

Universal Framework:

One single framework which can be used to create the full variability offunctions, specificities or properties which is originally sustained bya large collection of different frameworks, is called universalframework.

Binding of an Antibody to its Target:

The process which leads to a tight and specific association between anantibody and a corresponding molecule or ligand is called binding. Amolecule or ligand or any part of a molecukle or ligand which isrecognized by an antibody is called the target.

Replacing Genetic Sub-Sequences

A method by which an existing sub-sequence is removed using the flankingcleavage sites of this sub-sequence, and a new sub-sequence orcollection of sub-sequences, which contains ends compatible with thecleavage sites thus created, is inserted.

Assembling of Genetic Sequences:

Any process which is used to combine synthetic or natural geneticsequences in a specific manner in order to get longer genetic sequenceswhich contain at least parts of the used synthetic or natural geneticsequences.

Analysis of Homologous Genes:

The corresponding amino acid sequences of two or more genes are alignedto each other in a way which maximizes the correspondence betweenidentical or similar amino acid residues at all positions. These alignedsequences are termed homologous if the percentage of the Sum ofidentical and/or similar residues exceeds a defined threshold. Thisthreshold is commonly regarded by those skilled in the art as beingexceeded when at least 15 percent of the amino acids in the alignedgenes are identical, and at least 30 percent are similar.

LEGENDS TO FIGURES AND TABLES

FIG. 1: Flow chart outlining the process of construction of a synthetichuman antibody library based on consensus sequences.

FIG. 2: Alignment of consensus sequences designed for each subgroup(amino acid residues are shown with their standard one-letterabbreviation). (A) kappa sequences, (B) lambda sequences and (C), heavychain sequences. The positions are numbered according to Kabat (1991).In order to maximize homology in the alignment, gaps (−) have beenintroduced in the sequence at certain positions.

FIG. 3: Gene sequences of the synthetic V kappa consensus genes. Thecorresponding amino acid sequences (see FIG. 2) as well as the uniquecleavage sites are also shown.

FIG. 4: Gene sequences of the synthetic V lambda consensus genes. Thecorresponding amino acid sequences (see FIG. 2) as well as the uniquecleavage sites are also shown.

FIG. 5: Gene sequences of the synthetic V heavy chain consensus genes.The corresponding amino acid sequences (see FIG. 2) as well as theunique cleavage sites are also shown.

FIG. 6: Oligonucleotides used for construction of the consensus genes.The oligos are named according to the corresponding consensus gene, e.g.the gene Vκ1 was constructed using the six oligonucleotides O1K1 toO1K6. The oligonucleotides used for synthesizing the genes encoding theconstant domains Cκ (OCLK1 to 8) and CH1 (OCH1 to 8) are also shown.

FIG. 7A/B: Sequences of the synthetic genes encoding the constantdomains Cκ (A) and CH1 (B). The corresponding amino acid sequences aswell as unique cleavage sites introduced in these genes are also shown.

FIG. 7C: Functional map and sequence of module M24 comprising thesynthetic Cλ gene segment (huCL lambda).

FIG. 7D: Oligonucleotides used for synthesis of module M24.

FIG. 8: Sequence and restriction map of the synthetic gene encoding theconsensus single-chain fragment VH3-Vκ2. The signal sequence (aminoacids 1 to 21) was derived from the E. coli phoA gene (Skerra &Plückthun, 1988). Between the phoA signal sequence and the VH3 domain, ashort sequence stretch encoding 4 amino acid residues (amino acid 22 to25) has been inserted in order to allow detection of the single-chainfragment in Western blot or ELISA using the monoclonal antibody M1(Knappik & Plückthun, 1994). The last 6 basepairs of the sequence wereintroduced for cloning purposes (EcoRI site).

FIG. 9: Plasmid map of the vector pIG10.3 used for phage display of theH3κ2 scFv fragment. The vector is derived from pIG10 and contains thegene for the lac operon repressor, lacI, the artificial operon encodingthe H3κ2-gene3ss fusion under control of the lac promoter, the lppterminator of transcription, the single-strand replication origin of theE. coli phage f1 (F1_ORI), a gene encoding β-lactamase (bla) and theColEI derived origin of replication.

FIG. 10: Sequencing results of independent clones from the initiallibrary, translated into the corresponding amino acid sequences. (A)Amino acid sequence of the VH3 consensus heavy chain CDR3 (position 93to 102, Kabat numbering). (B) Amino acid sequences of 12 clones of the10-mer library. (C) Amino acid sequences of 11 clones of the 15-merlibrary, single base deletion.

FIG. 11: Expression test of individual library members. (A) Expressionof 9 independent clones of the 10-mer library. (B) Expression of 9independent clones of the 15-mer library. The lane designated with Mcontains the size marker. Both the gp3-scFv fusion and the scFv monomerare indicated.

FIG. 12: Enrichment of specific phage antibodies during the panningagainst FITC-BSA. The initial as well as the subsequentfluorescein-specific sub-libraries were panned against the blockingbuffer and the ratio of the phage eluted from the FITC-BSA coated wellvs. that from the powder milk coated well from each panning round ispresented as the “specificity factor”.

FIG. 13: Phage ELISA of 24 independent clones after the third round ofpanning tested for binding on FITC-BSA.

FIG. 14: Competition ELISA of selected FITC-BSA binding clones. TheELISA signals (OD_(405 nm)) of scFv binding without inhibition are takenas 100%.

FIG. 15: Sequencing results of the heavy chain CDR3s of independentclones after 3 rounds of panning against FITC-BSA, translated into thecorresponding amino acid sequences (position 93 to 102. Kabatnumbering).

FIG. 16: Coomassie-Blue stained SDS-PAGE of the purifiedanti-fluorescein scFv fragments: M: molecular weight marker, A: totalsoluble cell extract after induction, B: fraction of the flow-through,C, D and E: purified scFv fragments 1HA-3E4, 1HA-3E5 and 1HA-3E10,respectively.

FIG. 17: Enrichment of specific phage antibodies during the panningagainst β-estradiol-BSA, testosterone-BSA, BSA, ESL-1, interleukin-2,lymphotoxin-β, and LeY-BSA after three rounds of panning.

FIG. 18: ELISA of selected ESL-1 and β-estradiol binding clones.

FIG. 19: Selectivity and cross-reactivity of HuCAL antibodies: in thediagonal specific binding of HuCAL antibodies can be seen, off-diagonalsignals show non-specific cross-reactivity.

FIG. 20: Sequencing results of the heavy chain CDR3s of independentclones after 3 rounds of panning against β-estradiol-BSA, translatedinto the corresponding amino acid sequences (position 93 to 102, Kabatnumbering). One clone is derived from the 10mer library.

FIG. 21: Sequencing results of the heavy chain CDR3s of independentclones after 3 rounds of panning against testosterone-BSA, translatedinto the corresponding amino acid sequences (position 93 to 102, Kabatnumbering).

FIG. 22: Sequencing results of the heavy chain CDR3s of independentclones after 3 rounds of panning against lymphotoxin-β, translated intothe corresponding amino acid sequences (position 93 to 102, Kabatnumbering). One clone comprises a 14mer CDR, presumably introduced byincomplete coupling of the trinucleotide mixture during oligonucleotidesynthesis.

FIG. 23: Sequencing results of the heavy chain CDR3s of independentclones after 3 rounds of panning against ESL-1, translated into thecorresponding amino acid sequences (position 93 to 102, Kabatnumbering). Two clones are derived from the 10mer library. One clonecomprises a 16mer CDR, presumably introduced by chain elongation duringoligonucleotide synthesis using trinucleotides.

FIG. 24: Sequencing results of the heavy chain CDR3s of independentclones after 3 rounds of panning against BSA, translated into thecorresponding amino acid sequences (position 93 to 102, Kabatnumbering).

FIG. 25: Schematic representation of the modular pCAL vector system.

FIG. 25 a: List of restriction sites already used in or suitable for themodular HuCAL genes and pCAL vector system.

FIG. 26: List of the modular vector elements for the pCAL vector series:shown are only those restriction sites which are part of the modularsystem.

FIG. 27: Functional map and sequence of the multi-cloning site module(MCS).

FIG. 28: Functional map and sequence of the pMCS cloning vector series.

FIG. 29: Functional map and sequence of the pCAL module M1 (see FIG.26).

FIG. 30: Functional map and sequence of the pCAL module M7-III (see FIG.26).

FIG. 31: Functional map and sequence of the pCAL module M9-II (see FIG.26).

FIG. 32: Functional map and sequence of the pCAL module M11-II (see FIG.26).

FIG. 33: Functional map and sequence of the pCAL module M14-Ext2 (seeFIG. 26).

FIG. 34: Functional map and sequence of the PCAL module M17 (see FIG.26).

FIG. 35: Functional map and sequence of the modular vector pCAL4.

FIG. 35 a: Functional maps and sequences of additional pCAL modules (M2,M3, M7I, M7II, M8, M10II, M11II, M12, M13, M19, M20, M21, M41) and oflow-copy number plasmid vectors (pCALO1 to pCALO3).

FIG. 35 b: List of oligonucleotides and primers used for synthesis ofpCAL vector modules.

FIG. 36: Functional map and sequence of the β-lactamase cassette forreplacement of CDRs for CDR library cloning.

FIG. 37: Oligo and primer design for Vκ CDR3 libraries FIG. 38: Oligoand primer design for Vλ CDR3 libraries.

FIG. 39: Functional map of the pBS13 expression vector series.

FIG. 40: Expression of all 49 HuCAL scFvs obtained by combining each ofthe 7 VH genes with each of the 7 VL genes (pBS13, 30° C.); Values aregiven for the percentage of soluble vs. insoluble material, the totaland the soluble amount compared to the combination H3κ2, which was setto 100%. In addition, the corresponding values for the McPC603 scFv aregiven.

Table 1: Summary of human immunoglobulin germline sequences used forcomputing the germline membership of rearranged sequences. (A) kappasequences, (B) lambda sequences and (C), heavy chain sequences. (1) Thegermline name used in the various calculations, (2) the referencesnumber for the corresponding sequence (see appendix for sequence relatedcitations), (3) the family where each sequence belongs to and (4), thevarious names found in literature for germline genes with identicalamino acid sequences.

Table 2: Rearranged human sequences used for the calculation ofconsensus sequences. (A) kappa sequences, (B) lambda sequences and (C),heavy chain sequences. The table summarized the name of the sequence(1), the length of the sequence in amino acids (2), the germline family(3) as well as the computed germline counterpart (4). The number ofamino acid exchanges between the rearranged sequence and the germlinesequence is tabulated in (5), and the percentage of different aminoacids is given in (6). Column (7) gives the references number for thecorresponding sequence (see appendix for sequence related citations).

Table 3: Assignment of rearranged V sequences to their germlinecounterparts. (A) kappa sequences, (B) lambda sequences and (C), heavychain sequences. The germline genes are tabulated according to theirfamily (1), and the number of rearranged genes found for every germlinegene is given in (2).

Table 4: Computation of the consensus sequence of the rearranged V kappasequences. (A), V kappa subgroup 1, (B), V kappa subgroup 2, (C), Vkappa subgroup 3 and (D), V kappa subgroup 4. The number of each aminoacid found at each position is tabulated together with the statisticalanalysis of the data. (1) Amino acids are given with their standardone-letter abbreviations (and B means D or N, Z means E or Q and X meansany amino acid). The statistical analysis summarizes the number ofsequences found at each position (2), the number of occurrences of themost common amino acid (3), the amino acid residue which is most commonat this position (4), the relative frequency of the occurrence of themost common amino acid (5) and the number of different amino acids foundat each position (6).

Table 5: Computation of the consensus sequence of the rearranged Vlambda sequences. (A), V lambda subgroup 1, (B), V lambda subgroup 2,and (C), V lambda subgroup 3. The number of each amino acid found ateach position is tabulated together with the statistical analysis of thedata. Abbreviations are the same as in Table 4.

Table 6: Computation of the consensus sequence of the rearranged V heavychain sequences. (A), V heavy chain subgroup 1A, (B), V heavy chainsubgroup 1B, (C), V heavy chain subgroup 2, (D), V heavy chain subgroup3, (E), V heavy chain subgroup 4, (F), V heavy chain subgroup 5, and(G), V heavy chain subgroup 6. The number of each amino acid found ateach position is tabulated together with the statistical analysis of thedata. Abbreviations are the same as in Table 4.

EXAMPLES Example 1 Design of a Synthetic Human Combinatorial AntibodyLibrary (HuCAL)

The following example describes the design of a fully synthetic humancombinatorial antibody library (HuCAL), based on consensus sequences ofthe human immunoglobulin repertoire, and the synthesis of the consensusgenes. The general procedure is outlined in FIG. 1.

1.1 Sequence Database

1.1.1 Collection and Alignment of Human Immunoglobulin Sequences

In a first step, sequences of variable domains of human immunoglobulinshave been collected and divided into three sub bases: V heavy chain(VH), V kappa (Vκ) and V lambda (Vλ). For each sequence, the genesequence was then translated into the corresponding amino acid sequence.Subsequently, all amino acid sequences were aligned according to Kabatet al. (1991). In the case of Vλ sequences, the numbering system ofChuchana et al. (1990) was used. Each of the three main databases wasthen divided into two further sub bases: the first sub base containedall sequences derived from rearranged V genes, where more than 70positions of the sequence were known. The second sub base contained allgermline gene segments (without the D- and J-minigenes; pseudogenes withinternal stop codons were also removed). In all cases, where germlinesequences with identical amino acid sequence but different names werefound, only one sequence was used (see Table 1). The final databases ofrearranged sequences contained 386, 149 and 674 entries for Vκ, Vλ andVH, respectively. The final databases of germline sequences contained48, 26 and 141 entries for Vκ, Vλ and VH, respectively.

1.1.2 Assignment of Sequences to Subgroups

The sequences in the three germline databases where then groupedaccording to sequence homology (see also Tomlinson et al., 1992,Williams & Winter, 1993, and Cox et al., 1994). In the case of Vκ, 7families could be established. Vλ was divided into 8 families and VHinto 6 families. The VH germline genes of the VH7 family (Van Dijk etal., 1993) were grouped into the VH1 family, since the genes of the twofamilies are highly homologous. Each family contained different numbersof germline genes, varying from 1 (for example VH6) to 47 (VH3).

1.2 Analysis of Sequences

1.2.1 Computation of Germline Membership

For each of the 1209 amino acid sequences in the databases of rearrangedgenes, the nearest germline counterpart, i.e. the germline sequence withthe smallest number of amino acid differences was then calculated. Afterthe germline counterpart was found, the number of somatic mutationswhich occurred in the rearranged gene and which led to amino acidexchanges could be tabulated. In 140 cases, the germline counterpartcould not be calculated exactly, because more than one germline gene wasfound with an identical number of amino acid exchanges. These rearrangedsequences were removed from the database. In a few cases, the number ofamino acid exchanges was found to be unusually large (>20 for VL and >25for VH), indicating either heavily mutated rearranged genes orderivation from germline genes not present in the database. Since it wasnot possible to distinguish between these two possibilities, thesesequences were also removed from the database. Finally, 12 rearrangedsequences were removed from the database because they were found to havevery unusual CDR lengths and composition or unusual amino acids atcanonical positions (see below). In summary, 1023 rearranged sequencesout of 1209 (85%) could be clearly assigned to their germlinecounterparts (see Table 2).

After this calculation, every rearranged gene could be arranged in oneof the families established for the germline genes. Now the usage ofeach germline gene, i.e. the number of rearranged genes which originatefrom each germline gene, could be calculated (see Table 2). It was foundthat the usage was strongly biased towards a subset of germline genes,whereas most of the germline genes were not present as rearranged genesin the database and therefore apparently not used in the immune system(Table 3). This observation had already been reported in the case of Vκ(Cox, et al., 1994). All germline gene families, where no or only veryfew rearranged counterparts could be assigned, were removed from thedatabase, leaving 4 Vκ, 3 Vλ, and 6 VH families.

1.2.2 Analysis of CDR Conformations

The conformation of the antigen binding loops of antibody molecules, theCDRs, is strongly dependent on both the length of the CDRs and the aminoacid residues located at the so-called canonical positions (Chothia &Lesk, 1987). It has been found that only a few canonical structuresexist, which determine the structural repertoire of the immunoglobulinvariable domains (Chothia et al., 1989). The canonical amino acidpositions can be found in CDR as well as framework regions. The 13 usedgermline families defined above (7 VL and 6 VH) were now analyzed fortheir canonical structures in order to define the structural repertoireencoded in these families.

In 3 of the 4 Vκ families (Vκ1, 2 and 4), one different type of CDR1conformation could be defined for every family. The family Vκ3 showedtwo types of CDR1 conformation: one type which was identical to Vκ1 andone type only found in Vκ3. All Vκ CDR2s used the same type of canonicalstructure. The CDR3 conformation is not encoded in the germline genesegments. Therefore, the 4 Vκ families defined by sequence homology andusage corresponded also to 4 types of canonical structures found in Vκgermline genes.

The 3 Vλ families defined above showed 3 types of CDR1 conformation,each family with one unique type. The Vλ1 family contained 2 differentCDR1 lengths (13 and 14 amino acids), but identical canonical residues,and it is thought that both lengths adopt the same canonicalconformation (Chothia & Lesk, 1987). In the CDR2 of the used Vλgermlines, only one canonical conformation exists, and the CDR3conformation is not encoded in the germline gene segments. Therefore,the 3 Vλ families defined by sequence homology and usage correspondedalso to 3 types of canonical structures.

The structural repertoire of the human VH sequences was analyzed indetail by Chothia et al., 1992. In total, 3 conformations of CDR1 (H1-1,H1-2 and H1-3) and 6 conformations of CDR2 (H2-1, H2-2, H2-3, H2-4, H2-5and H2-x) could be defined. Since the CDR3 is encoded in the D- andJ-minigene segments, no particular canonical residues are defined forthis CDR.

All the members of the VH1 family defined above contained the CDR1conformation H1-1, but differed in their CDR2 conformation: the H2-2conformation was found in 6 germline genes, whereas the conformationH2-3 was found in 8 germline genes. Since the two types of CDR2conformations are defined by different types of amino acid at theframework position 72, the VH1 family was divided into two subfamilies:VH1A with CDR2 conformation H2-2 and VH1B with the conformation H2-3 Themembers of the VH2 family all had the conformations H1-3 and H2-1 inCDR1 and CDR2, respectively. The CDR1 conformation of the VH3 memberswas found in all cases to be H1-1, but 4 different types were found inCDR2 (H2-1, H2-3, H2-4 and H2-x). In these CDR2 conformations, thecanonical framework residue 71 is always defined by an arginine.Therefore, it was not necessary to divide the VH3 family intosubfamilies, since the 4 types of CDR2 conformations were defined solelyby the CDR2 itself. The same was true for the VH4 family. Here, all 3types of CDR1 conformations were found, but since the CDR1 conformationwas defined by the CDR itself (the canonical framework residue 26 wasfound to be glycine in all cases), no subdivisions were necessary. TheCDR2 conformation of the VH4 members was found to be H2-1 in all cases.All members of the VH5 family were found to have the conformation H1-1and H2-2, respectively. The single germline gene of the VH6 family hadthe conformations H1-3 and H2-5 in CDR1 and CDR2, respectively.

In summary, all possible CDR conformations of the Vκ and Vλ genes werepresent in the 7 families defined by sequence comparison. From the 12different CDR conformations found in the used VH germline genes, 7 couldbe covered by dividing the family VH1 into two subfamilies, therebycreating 7 VH families. The remaining 5 CDR conformations (3 in the VH3and 2 in the VH4 family) were defined by the CDRs themselves and couldbe created during the construction of CDR libraries. Therefore, thestructural repertoire of the used human V genes could be covered by 49(7×7) different frameworks.

1.2.3 Computation of Consensus Sequences

The 14 databases of rearranged sequences (4 Vκ, 3 Vλ, and 7 VH) wereused to compute the HuCAL consensus sequences of each subgroup (4HuCAL-Vκ, 3 HuCAL-Vλ, 7 HuCAL-VH, see Table 4, 5 and 6). This was doneby counting the number of amino acid residues used at each position(position variability) and subsequently identifying the amino acidresidue most frequently used at each position. By using the rearrangedsequences instead of the used germline sequences for the calculation ofthe consensus, the consensus was weighted according to the frequency ofusage. Additionally, frequently mutated and highly conserved positionscould be identified. The consensus sequences were cross-checked with theconsensus of the germline families to see whether the rearrangedsequences were biased at certain positions towards amino acid residueswhich do not occur in the collected germline sequences, but this wasfound not to be the case. Subsequently, the number of differences ofeach of the 14 consensus sequences to each of the germline sequencesfound in each specific family was calculated. The overall deviation fromthe most homologous germline sequence was found to be 2.4 amino acidresidues (s.d.=2.7), ensuring that the “artificial” consensus sequencescan still be considered as truly human sequences as far asimmunogenicity is concerned.

1.3 Structural Analysis

So far, only sequence information was used to design the consensussequences. Since it was possible that during the calculation certainartificial combinations of amino acid residues have been created, whichare located far away in the sequence but have contacts to each other inthe three dimensional structure, leading to destabilized or evenmisfolded frameworks, the 14 consensus sequences were analyzed accordingto their structural properties.

It was rationalized that all rearranged sequences present in thedatabase correspond to functional and therefore correctly foldedantibody molecules. Hence, the most homologous rearranged sequence wascalculated for each consensus sequence. The positions where theconsensus differed from the rearranged sequence were identified aspotential “artificial residues” and inspected.

The inspection itself was done in two directions. First, the localsequence stretch around each potentially “artificial residue” wascompared with the corresponding stretch of all the rearranged sequences.If this stretch was found to be truly artificial, i.e. never occurred inany of the rearranged sequences, the critical residue was converted intothe second most common amino acid found at this position and analyzedagain. Second, the potentially “artificial residues” were analyzed fortheir long range interactions. This was done by collecting all availablestructures of human antibody variable domains from the corresponding PDBfiles and calculating for every structure the number and type ofinteractions each amino acid residue established to each side-chain.These “interaction maps” were used to analyze the probableside-chain/side-chain interactions of the potentially “artificialresidues”. As a result of this analysis, the following residues wereexchanged (given is the name of the gene, the position according toKabat's numbering scheme, the amino acid found at this position as themost abundant one and the amino acid which was used instead):

-   VH2: S₆₅T-   Vκ1: N₃₄A,-   Vκ3: G₉A, D₆₀A, R₇₇S-   Vλ3: V₇₈T    1.4 Design of CDR Sequences

The process described above provided the complete consensus sequencesderived solely from the databases of rearranged sequences. It wasrationalized that the CDR1 and CDR2 regions should be taken from thedatabases of used germline sequences, since the CDRs of rearranged andmutated sequences are biased towards their particular antigens.Moreover, the germline CDR sequences are known to allow binding to avariety of antigens in the primary immune response, where only CDR3 isvaried. Therefore, the consensus CDRs obtained from the calculationsdescribed above were replaced by germline CDRs in the case of VH and Vκ.In the case of Vλ, a few amino acid exchanges were introduced in some ofthe chosen germline CDRs in order to avoid possible protease cleavagesites as well as possible structural constraints.

The CDRs of following germline genes have been chosen: HuCAL gene CDR1CDR2 HuCAL-VH1A VH1-12-1 VH1-12-1 HuCAL-VH1B VH1-13-16 VH1-13-6, -7, -8,-9 HuCAL-VH2 VH2-31-10, -11, VH2-31-3, -4 -12, -13 HuCAL-VH3 VH3-13-8,-9, -10 VH3-13-8, -9, -10 HuCAL-VH4 VH4-11-7 to -14 VH4-11-8, -9, -11,-12, -14, -16 VH4-31-17, -18, -19, -20 HuCAL-VH5 VH5-12-1, -2 VH5-12-1,-2 HuCAL-VH6 VH6-35-1 VH6-35-1 HuCAL-Vκ1 Vκ1-14, -15 Vκ1-2, -3, -4, -5,-7, -8, -12, -13, -18, -19 HuCAL-Vκ2 Vκ2-6 Vκ2-6 HuCAL-Vκ3 Vκ3-1, -4Vκ3-4 HuCAL-Vκ4 Vκ4-1 Vκ4-1 HuCAL-Vλ1 HUMLV117, DPL5 DPL5 HuCAL-Vλ2DPL11, DPL12 DPL12 HuCAL-Vλ3 DPL23 HUMLV318

In the case of the CDR3s, any sequence could be chosen since these CDRswere planned to be the first to be replaced by oligonucleotidelibraries. In order to study the expression and folding behavior of theconsensus sequences in E. coli, it would be useful to have all sequenceswith the same CDR3, since the influence of the CDR3s on the foldingbehavior would then be identical in all cases. The dummy sequencesQQHYTTPP and ARWGGDGFYAMDY were selected for the VL chains (kappa andlambda) and for the VH chains, respectively. These sequences are knownto be compatible with antibody folding in E. coli (Carter et al., 1992).

1.5 Gene Design

The final outcome of the process described above was a collection of 14HuCAL amino acid sequences, which represent the frequently usedstructural antibody repertoire of the human immune system (see FIG. 2).These sequences were back-translated into DNA sequences. In a firststep, the back-translation was done using only codons which are known tobe frequently used in E. coli. These gene sequences were then used forcreating a database of all possible restriction endonuclease sites,which could be introduced without changing the corresponding amino acidsequences. Using this database, cleavage sites were selected which werelocated at the flanking regions of all sub-elements of the genes (CDRsand framework regions) and which could be introduced in all HuCAL VH, Vκor Vλ genes simultaneously at the same position. In a few cases it wasnot possible to find cleavage sites for all genes of a subgroup. Whenthis happened, the amino acid sequence was changed, if this was possibleaccording to the available sequence and structural information. Thisexchange was then analyzed again as described above. In total, thefollowing 6 amino acid residues were exchanged during this design (givenis the name of the gene, the position according to Kabat's numberingscheme, the amino acid found at this position as the most abundant oneand the amino acid which was used instead):

-   VH2: T₃Q-   VH6: S₄₂G-   Vκ3: E₁D, I₅₈V-   Vκ4: K₂₄R-   Vλ3: T₂₂S

In one case (5′-end of VH framework 3) it was not possible to identify asingle cleavage site for all 7 VH genes. Two different type of cleavagesites were used instead: BstEII for HuCAL VH1A, VH1B, VH4 and VH5, andNspV for HuCAL VH2, VH3, VH4 and VH6.

Several restriction endonuclease sites were identified, which were notlocated at the flanking regions of the sub-elements but which could beintroduced in every gene of a given group without changing the aminoacid sequence. These cleavage sites were also introduced in order tomake the system more flexible for further improvements. Finally, all butone remaining restriction endonuclease sites were removed in every genesequence. The single cleavage site, which was not removed was differentin all genes of a subgroup and could be therefore used as a“fingerprint” site to ease the identification of the different genes byrestriction digest. The designed genes, together with the correspondingamino acid sequences and the group-specific restriction endonucleasesites are shown in FIGS. 3, 4 and 5, respectively.

1.6 Gene Synthesis and Cloning

The consensus genes were synthesized using the method described byProdromou & Pearl, 1992, using the oligonucleotides shown in FIG. 6.Gene segments encoding the human constant domains Cκ, Cλ and CH1 werealso synthesized, based on sequence information given by Kabat et al.,1991 (see FIG. 6 and FIG. 7). Since for both the CDR3 and the framework4 gene segments identical sequences were chosen in all HuCAL Vκ, Vλ andVH genes, respectively, this part was constructed only once, togetherwith the corresponding gene segments encoding the constant domains. ThePCR products were cloned into pCR-Script KS(+) (Stratagene, Inc.) orpZErO-1 (Invitrogen, Inc.) and verified by sequencing.

Example 2 Cloning and Testing of a HuCAL-Based Antibody Library

A combination of two of the synthetic consensus genes was chosen afterconstruction to test whether binding antibody fragments can be isolatedfrom a library based on these two consensus frameworks. The two geneswere clones as a single-chain Fv (scFv) fragment, and a VH-CDR3 librarywas inserted. In order to test the library for the presence offunctional antibody molecules, a selection procedure was carried outusing the small hapten fluorescein bound to BSA (FITC-BSA) as antigen.

2.1 Cloning of the HuCAL VH3-Vk2 scFv Fragment

In order to test the design of the consensus genes, one randomly chosencombination of synthetic light and heavy gene (HuCAL-Vκ2 and HuCAL-VH3)was used for the construction of a single-chain antibody (scFv)fragment. Briefly, the gene segments encoding the VH3 consensus gene andthe CH1 gene segment including the CDR3-framework 4 region, as well asthe Vκ2 consensus gene and the Cκ gene segment including theCDR3-framework 4 region were assembled yielding the gene for the VH3-CH1Fd fragment and the gene encoding the Vκ2-Cκ light chain, respectively.The CH1 gene segment was then replaced by an oligonucleotide cassetteencoding a 20-mer peptide linker with the sequence AGGGSGGGGSGGGGSGGGGS.The two oligonucleotides encoding this linker were5′-TCAGCGGGTGGCGGTTCTGGCGGCGGTGGGAGCGGTGGCGGTGGTTCTGGCGGTGGTGGTTCCGATATCGGTCCACGTACGG-3′and 5′-AATTCCGTACGTGGACCGATATCGGAACCACCACCGCCAGAACCACCGCCACCGCTCCCACCGCCGCCAGAACCGCCACCCGC-3′, respectively. Finally, the HuCAL-Vκ2 gene wasinserted via EcoRV and BsiWI into the plasmid encoding theHuCAL-VH3-linker fusion, leading to the final gene HuCAL-VH3-Vκ2, whichencoded the two consensus sequences in the single-chain formatVH-linker-VL. The complete coding sequence is shown in FIG. 8.

2.2 Construction of a Monovalent Phage-Display Phagemid Vector pIG10.3

Phagemid pIG10.3 (FIG. 9) was constructed in order to create aphage-display system (Winter et al., 1994) for the H3κ2 scFv gene.Briefly, the EcoRI/HindIII restriction fragment in the phagemid vectorpIG10 (Ge et al., 1995) was replaced by the c-myc followed by an ambercodon (which encodes an glutamate in the amber-suppresser strain XL1Blue and a stop codon in the non-suppresser strain JM83) and a truncatedversion of the gene III (fusion junction at codon 249, see Lowman etal., 1991) through PCR mutagenesis.

2.3 Construction of H-CDR3 Libraries

Heavy chain CDR3 libraries of two lengths (10 and 15 amino acids) wereconstructed using trinucleotide codon containing oligonucleotides(Virnekäs et al., 1994) as templates and the oligonucleotidescomplementing the flanking regions as primers. To concentrate only onthe CDR3 structures that appear most often in functional antibodies, wekept the salt-bridge of R_(H94) and D_(H)101 in the CDR3 loop. For the15-mer library, both phenylalanine and methionine were introduced atposition 100 since these two residues were found to occur quite often inhuman CDR3s of this length (not shown). For the same reason, valine andtyrosine were introduced at position 102. All other randomized positionscontained codons for all amino acids except cystein, which was not usedin the trinucleotide mixture.

The CDR3 libraries of lengths 10 and 15 were generated from the PCRfragments using oligonucleotide templates O3HCDR103T.(5′-GATACGGCCGTGTATTATTGCGCGCGT (TRI)₆GATTATTGGGGCCMGGCACCCTG-3′) andO3HCDR153T (5′-GATACGGCCGTGTATTATTGCGCGCGT(TRI)₁₀(TTT/ATG)GAT(GTT/TAT)TGGGGCCAAGGCACCCTG-3′), andprimers O3HCDR35 (5′-GATACGGCCGTGTATTATTGC-3′) and O3HCDR33(5′-CAGGGTGCCTTGGCCCC-3′), where TRI are trinucleotide mixturesrepresenting all amino acids without cystein, (TTT/ATG) and (GTT/TAT)are trinucleotide mixtures encoding the amino acidsphenylalanine/methionine and valine/tyrosine, respectively. Thepotential diversity of these libraries was 4.7×10⁷ and 3.4×10¹⁰ for10-mer and 15-mer library, respectively. The library cassettes werefirst synthesized from PCR amplification of the oligo templates in thepresence of both primers: 25 pmol of the oligo template O3HCDR103T orO3HCDR153T, 50 pmol each of the primers O3HCDR35 and O3HCDR33, 20 nmolof dNTP, 10× buffer and 2.5 units of Pfu DNA polymerase (Stratagene) ina total volume of 100 μl for 30 cycles (1 minute at 92° C., 1 minute at62° C. and 1 minute at 72° C.). A hot-start procedure was used. Theresulting mixtures were phenol-extracted, ethanol-precipitated anddigested overnight with EagI and StyI. The vector pIG10.3-sCH3κ2cat,where the EagI-StyI fragment in the vector pIG10.3-sCH3κ2 encoding theH-CDR3 was replaced by the chloramphenicol acetyltransferase gene (cat)flanked with these two sites, was similarly digested. The digestedvector (35 μg) was gel-purified, and ligated with 100 μg of the librarycassette overnight at 16° C. The ligation mixtures were isopropanolprecipitated, air-dried and the pellets were redissolved in 100 μl ofddH2O. The ligation was mixed with 1 ml of freshly preparedelectrocompetent XL1 Blue on ice. 20 rounds of electroporation wereperformed and the transformants were diluted in SOC medium, shaken at37° C. for 30 minutes and plated out on large LB plates(Amp/Tet/Glucose) at 37° C. for 6-9 hrs. The number of transformants(library size) was 3.2×10⁷ and 2.3×10⁷ for the 10-mer and the 15-merlibrary, respectively. The colonies were suspended in 2×YT medium(Amp/Tet/Glucose) and stored as glycerol culture.

In order to test the quality of the initial library, phagemids from 24independent colonies (12 from the 10-mer and 12 from the 15-mer library,respectively) were isolated and analyzed by restriction digestion andsequencing. The restriction analysis of the 24 phagemids indicated thepresence of intact vector in all cases. Sequence analysis of theseclones (see FIG. 10) indicated that 22 out of 24 contained a functionalsequence in their heavy chain CDR3 regions. 1 out of 12 clones of the10-mer library had a CDR3 of length 9 instead of 10, and 2 out of 12clones of the 15-mer library had no open reading frame, thereby leadingto a non-functional scFv; one of these two clones contained twoconsecutive inserts, but out of frame (data not shown). All codonsintroduced were presented in an even distribution.

Expression levels of individual library members were also measured.Briefly, 9 clones from each library were grown in 2×YT medium containingAmp/Tet/0.5% glucose at 37° C. overnight. Next day, the cultures werediluted into fresh medium with Amp/Tet. At an OD_(600 nm) of 0.4, thecultures were induced with 1 mM of IPTG and shaken at RT overnight. Thenthe cell pellets were suspended in 1 ml of PBS buffer+1 mM of EDTA. Thesuspensions were sonicated and the supernatants were separated on anSDS-PAGE under reducing conditions, blotted on nylon membrane anddetected with anti-FLAG M1 antibody (see FIG. 11). From the nine clonesof the 10-mer library, all express the scFv fragments. Moreover, thegene III/scFv fusion proteins were present in all cases. Among the nineclones from the 15-mer library analyzed, 6/9 (67%) led to the expressionof both scFv and the gene III/scFv fusion proteins. More importantly,all clones expressing the scFvs and gene III/scFv fusions gave rise toabout the same level of expression.

2.4 Biopanning

Phages displaying the antibody libraries were prepared using standardprotocols. Phages derived from the 10-mer library were mixed with phagesfrom the 15-mer library in a ratio of 20:1 (1×10¹⁰ cfu/well of the10-mer and 5×10⁸ cfu/well of the 15-mer phages, respectively).Subsequently, the phage solution was used for panning in ELISA plates(Maxisorp, Nunc) coated with FITC-BSA (Sigma) at concentration of 100μg/ml in PBS at 4° C. overnight. The antigen-coated wells were blockedwith 3% powder milk in PBS and the phage solutions in 1% powder milkwere added to each well and the plate was shaken at RT for 1 hr. Thewells were then washed with PBST and PBS (4 times each with shaking atRT for 5 minutes). The bound phages were eluted with 0.1 M triethylamine(TEA) at RT for 10 minutes. The eluted phage solutions were immediatelyneutralized with ½ the volume of 1 M Tris Cl, pH 7.6. Eluted phagesolutions (ca. 450 μl) were used to infect 5 ml of XL1 Blue cells at 37°C. for 30 min. The infected cultures were then plated out on large LBplates (Amp/Tet/Glucose) and allowed to grow at 37° C. until thecolonies were visible. The colonies were suspended in 2×YT medium andthe glycerol cultures were made as above described. This panning roundwas repeated twice, and in the third round elution was carried out withaddition of fluorescein in a concentration of 100 μg/ml in PBS. Theenrichment of specific phage antibodies was monitored by panning theinitial as well as the subsequent fluorescein-specific sub-librariesagainst the blocking buffer (FIG. 12). Antibodies with specificityagainst fluorescein were isolated after 3 rounds of panning.

2.5 ELISA Measurements

One of the criteria for the successful biopanning is the isolation ofindividual phage clones that bind to the targeted antigen or hapten. Weundertook the isolation of anti-FITC phage antibody clones andcharacterized them first in a phage ELISA format. After the 3rd round ofbiopanning (see above), 24 phagemid containing clones were used toinoculate 100 μl of 2×YT medium (Amp/Tet/Glucose) in an ELISA plate(Nunc), which was subsequently shaken at 37° C. for 5 hrs. 100 μl of2×YT medium (Amp/Tet/1 mM IPTG) were added and shaking was continued for30 minutes. A further 100 μl of 2×YT medium (Amp/Tet) containing thehelper phage (1×10⁹ cfu/well) was added and shaking was done at RT for 3hrs. After addition of kanamycin to select for successful helper phageinfection, the shaking was continued overnight. The plates were thencentrifuged and the supernatants were pipetted directly into ELISA wellscoated with 100 μl FITC-BSA (100 μg/ml) and blocked with milk powder.Washing was performed similarly as during the panning procedure and thebound phages were detected with anti-M13 antibody-POD conjugate(Pharmacia) using soluble POD substrate (Boehringer-Mannheim). Of the 24clones screened against FITC-BSA, 22 were active in the ELISA (FIG. 13).The initial libraries of similar titer gave rise to no detectablesignal.

Specificity for fluorescein was measured in a competitive ELISA.Periplasmic fractions of five FITC specific scFvs were prepared asdescribed above. Western blotting indicated that all clones expressedabout the same amount of scFv fragment (data not shown). ELISA wasperformed as described above, but additionally, the periplasmicfractions were incubated 30 min at RT either with buffer (noinhibition), with 10 mg/ml BSA (inhibition with BSA) or with 10 mg/mlfluorescein (inhibition with fluorescein) before adding to the well.Binding scFv fragment was detected using the anti-FLAG antibody M1. TheELISA signal could only be inhibited, when soluble fluorescein wasadded, indicating binding of the scFvs was specific for fluorescein(FIG. 14).

2.6 Sequence Analysis

The heavy chain CDR3 region of 20 clones were sequenced in order toestimate the sequence diversity of fluorescein binding antibodies in thelibrary (FIG. 15). In total, 16 of 20 sequences (80%) were different,showing that the constructed library contained a highly diverserepertoire of fluorescein binders. The CDR3s showed no particularsequence homology, but contained on average 4 arginine residues. Thisbias towards arginine in fluorescein binding antibodies had already beendescribed by Barbas et al., 1992.

2.7 Production

E. coli JM83 was transformed with phagemid DNA of 3 selected clones andcultured in 0.5 L 2×YT medium. Induction was carried out with 1 mM IPTGat OD_(600 nm)=0.4 and growth was continued with vigorous shaking at RTovernight. The cells were harvested and pellets were suspended in PBSbuffer and sonicated. The supernatants were separated from the celldebris via centrifugation and purified via the BioLogic system (Bio-Rad)by with a POROS®MC 20 column (IMAC, PerSeptive Biosystems, Inc.) coupledwith an ion-exchange chromatography column. The ion-exchange column wasone of the POROS®HS, CM or HQ or PI 20 (PerSeptive Biosystems, Inc.)depended on the theoretical pI of the scFv being purified. The pH of allthe buffers was adjusted to one unit lower or higher than the pI of thescFv being purified throughout. The sample was loaded onto the firstIMAC column, washed with 7 column volumes of 20 mM sodium phosphate, 1 MNaCl and 10 mM imidazole. This washing was followed by 7 column volumesof 20 mM sodium phosphate and 10 mM imidazole. Then 3 column volumes ofan imidazole gradient (10 to 250 mM) were applied and the eluent wasconnected directly to the ion-exchanger. Nine column volumes ofisocratic washing with 250 mM imidazole was followed by 15 columnvolumes of 250 mM to 100 mM and 7 column volumes of an imidazole/NaClgradient (100 to 10 mM imidazole, 0 to 1 M NaCl). The flow rate was 5ml/min. The purity of scFv fragments was checked by SDS-PAGE Coomassiestaining (FIG. 16). The concentration of the fragments was determinedfrom the absorbance at 280 nm using the theoretically determinedextinction coefficient (Gill & von Hippel, 1989). The scFv fragmentscould be purified to homogeneity (see FIG. 16). The yield of purifiedfragments ranged from 5 to 10 mg/L/OD.

Example 3 HuCAL H3κ2 Library Against a Collection of Antigens

In order to test the library used in Example 2 further, a new selectionprocedure was carried out using a variety of antigens comprisingβ-estradiol, testosterone, Lewis-Y epitope (LeY), interleukin-2 (IL-2),lymphotoxin-β (LT-β), E-selectin ligand-1 (ESL-1), and BSA.

3.1 Biopanning

The library and all procedures were identical to those described inExample 2. The ELISA plates were coated with β-estradiol-BSA (100μg/ml), testosterone-BSA (100 μg/ml), LeY-BSA (20 μg/ml) IL-2 (20μg/ml), ESL-1 (20 μg/ml) and BSA (100 μg/ml), LT-β (denatured protein,20 μg/ml). In the first two rounds, bound phages were eluted with 0.1 Mtriethylamine (TEA) at RT for 10 minutes. In the case of BSA, elutionafter three rounds of panning was carried out with addition of BSA in aconcentration of 100 μg/ml in PBS. In the case of the other antigens,third round elution was done with 0.1 M triethylamine. In all casesexcept LeY, enrichment of binding phages could be seen (FIG. 17).Moreover, a repetition of the biopanning experiment using only the15-mer library resulted in the enrichment of LeY-binding phages as well(data not shown).

3.2. ELISA Measurements

Clones binding to β-estradiol, testosterone, LeY, LT-β, ESL-1 and BSAwere further analyzed and characterized as described in Example 2 forFITC. ELISA data for anti-β-estradiol and anti-ESL-1 antibodies areshown in FIG. 18. In one experiment, selectivity and cross-reactivity ofbinding scFv fragments were tested. For this purpose, an ELISA plate wascoated with FITC, testosterone, β-estradiol, BSA, and ESL-1, with 5wells for each antigen arranged in 5 rows, and 5 antibodies, one againsteach of the antigens, were screened against each of the antigens. FIG.19 shows the specific binding of the antibodies to the antigen it wasselected for, and the low cross-reactivity with the other four antigens.

3.3 Sequence Analysis

The sequencing data of several clones against β-estradiol (34 clones),testosterone (12 clones), LT-β (23 clones), ESL-1 (34 clones), and BSA(10 clones) are given in FIGS. 20 to 24.

Example 4 Vector Construction

To be able to take advantage of the modularity of the consensus generepertoire, a vector system had to be constructed which could be used inphage display screening of HuCAL libraries and subsequent optimizationprocedures. Therefore, all necessary vector elements such as origins ofsingle-stranded or double-stranded replication, promotor/operator,repressor or terminator elements, resistance genes, potentialrecombination sites, gene III for display on filamentous phages, signalsequences, or detection tags had to be made compatible with therestriction site pattern of the modular consensus genes. FIG. 25 shows aschematic representation of the pCAL vector system and the arrangementof vector modules and restriction sites therein. FIG. 25 a shows a listof all restriction sites which are already incorporated into theconsensus genes or the vector elements as part of the modular system orwhich are not yet present in the whole system. The latter could be usedin a later stage for the introduction of or within new modules.

4.1 Vector Modules

A series of vector modules was constructed where the restriction sitesflanking the gene sub-elements of the HuCAL genes were removed, thevector modules themselves being flanked by unique restriction sites.These modules were constructed either by gene synthesis or bymutagenesis of templates. Mutagenesis was done by add-on PCR, bysite-directed mutagenesis (Kunkel et al., 1991) or multisiteoligonucleotide-mediated mutagenesis (Sutherland et al., 1995; Perlak,1990) using a PCR-based assembly method.

FIG. 26 contains a list of the modules constructed. Instead of theterminator module M9 (HindIII-Ipp-PacI), a larger cassette M9II wasprepared to introduce FseI as additional restriction site. M9II can becloned via HindIII/BsrGI.

All vector modules were characterized by restriction analysis andsequencing. In the case of module M11-II, sequencing of the modulerevealed a two-base difference in positions 164/65 compared to thesequence database of the template. These two different bases (CA→GC)created an additional BanII site. Since the same two-base differenceoccurs in the f1 origin of other bacteriophages, it can be assumed thatthe two-base difference was present in the template and not created bymutagenesis during cloning. This BanII site was removed by site-directedmutagenesis, leading to module M11-II. The BssSI site of module M14could initially not be removed without impact on the function of theColE1 origin, therefore M14-Ext2 was used for cloning of the first pCALvector series. FIGS. 29 to 34 are showing the functional maps andsequences of the modules used for assembly of the modular vector pCAL4(see below). The functional maps and sequences of additional modules canbe found in FIG. 35 a. FIG. 35 b contains a list of oligonucleotides andprimers used for the synthesis of the modules.

4.2 Cloning Vector pMCS

To be able to assemble the individual vector modules, a cloning vectorpMCS containing a specific multi-cloning site (MCS) was constructed.First, an MCS cassette (FIG. 27) was made by gene synthesis. Thiscassette contains all those restriction sites in the order necessary forthe sequential introduction of all vector modules and can be cloned viathe 5′-HindIII site and a four base overhang at the 3′-end compatiblewith an AatII site. The vector pMCS (FIG. 28) was constructed bydigesting pUC19 with AatII and HindIII, isolating the 2174 base pairfragment containing the bla gene and the ColE1 origin, and ligating theMCS cassette.

4.3 Cloning of Modular Vector pCAL4

This was cloned step by step by restriction digest of pMCS andsubsequent ligation of the modules M1 (via AatII/XbaI), M7III (viaEcoRI/HindIII), and M9II (via HindIII/BsrGI), and M11-II (viaBsrGI/NheI). Finally, the bla gene was replaced by the cat gene moduleM17 (via AatII/BglII), and the wild type ColE1 origin by module M14-Ext2(via BglII/NheI). FIG. 35 is showing the functional map and the sequenceof pCAL4.

4.4 Cloning of Low-Copy Number Plasmid Vectors pCALO

A series of low-copy number plasmid vectors was constructed in a similarway using the p15A module M12 instead of the ColE1 module M14-Ext2. FIG.35 a is showing the functional maps and sequences of the vectors pCALO1to pCALO3.

Example 5 Construction of a HuCAL scFv Library

5.1. Cloning of All 49 HuCAL scFv Fragments

All 49 combinations of the 7 HuCAL-VH and 7 HuCAL-VL consensus geneswere assembled as described for the HuCAL VH3-Vκ2 scFv in Example 2 andinserted into the vector pBS12, a modified version of the pLisc seriesof antibody expression vectors (Skerra et al., 1991).

5.2 Construction of a CDR Cloning Cassette

For replacement of CDRs, a universal β-lactamase cloning cassette wasconstructed having a multi-cloning site at the 5′-end as well as at the3′-end. The 5′-multi-cloning site comprises all restriction sitesadjacent to the 5′-end of the HuCAL VH and VL CDRs, the 3′-multi-cloningsite comprises all restriction sites adjacent to the 3′ end of the HuCALVH and VL CDRs. Both 5′- and 3′-multi-cloning site were prepared ascassettes via add-on PCR using synthetic oligonucleotides as 5′- and3′-primers using wild type β-lactamase gene as template. FIG. 36 showsthe functional map and the sequence of the cassette bla-MCS.

5.3. Preparation of VL-CDR3 Library Cassettes

The VL-CDR3 libraries comprising 7 random positions were generated fromthe PCR fragments using oligonucleotide templates Vκ1&Vκ3, Vκ2 and Vκ4and primers O_K3L_(—)5 and O_K3L_(—)3 (FIG. 37) for the Vκ genes, and Vλand primers O_L3L_(—)5 (5′-GCAGAAGGCGAACGTCC-3′) and O_L3LA_(—)3 (FIG.38) for the Vλ genes. Construction of the cassettes was performed asdescribed in Example 2.3.

5.4 Cloning of HuCAL scFv Genes With VL-CDR3 Libraries

Each of the 49 single-chains was subcloned into pCAL4 via XbaI/EcoRI andthe VL-CDR3 replaced by the β-lactamase cloning cassette via BbsI/MscI,which was then replaced by the corresponding VL-CDR3 library cassettesynthesized as described above. This CDR replacement is described indetail in Example 2.3 where the cat gene was used.

5.5 Preparation of VH-CDR3 Library Cassette

The VH-CDR3 libraries were designed and synthesized as described inExample 2.3.

5.6 Cloning of HuCAL scFv Genes With VL- and VH-CDR3 Libraries

Each of the 49 single-chain VL-CDR3 libraries was digested withBssHII/StyI to replace VH-CDR3. The “dummy” cassette digested withBssHII/StyI was inserted, and was then replaced by a correspondingVH-CDR3 library cassette synthesized as described above.

Example 6 Expression Tests

Expression and toxicity studies were performed using the scFv formatVH-linker-VL. All 49 combinations of the 7 HuCAL-VH and 7 HuCAL-VLconsensus genes assembled as described in Example 5 were inserted intothe vector pBS13, a modified version of the pLisc series of antibodyexpression vectors (Skerra et al., 1991). A map of this vector is shownin FIG. 39.

E. coli JM83 was transformed 49 times with each of the vectors andstored as glycerol stock. Between 4 and 6 clones were testedsimultaneously, always including the clone H3κ2, which was used asinternal control throughout. As additional control, the McPC603 scFvfragment (Knappik & Plückthun, 1995) in pBS13 was expressed underidentical conditions. Two days before the expression test was performed,the clones were cultivated on LB plates containing 30 μg/mlchloramphenicol and 60 mM glucose. Using this plates an 3 ml culture (LBmedium containing 90 μg chloramphenicol and 60 mM glucose) wasinoculated overnight at 37° C. Next day the overnight culture was usedto inoculate 30 ml LB medium containing chloramphenicol (30 μg/ml). Thestarting OD_(600 nm) was adjusted to 0.2 and a growth temperature of 30°C. was used. The physiology of the cells was monitored by measuringevery 30 minutes for 8 to 9 hours the optical density at 600 nm. Afterthe culture reached an OD_(600 nm) of 0.5, antibody expression wasinduced by adding IPTG to a final concentration of 1 mM. A 5 ml aliquotof the culture was removed after 2 h of induction in order to analyzethe antibody expression. The cells were lysed and the soluble andinsoluble fractions of the crude extract were separated as described inKnappik & Plückthun, 1995. The fractions were assayed by reducingSDS-PAGE with the samples normalized to identical optical densities.After blotting and immunostaining using the α-FLAG antibody M1 as thefirst antibody (see Ge et al., 1994) and an Fc-specific anti-mouseantiserum conjugated to alkaline phosphatase as the second antibody, thelanes were scanned and the intensities of the bands of the expected size(appr. 30 kDa) were quantified densitometrically and tabulated relativeto the control antibody (see FIG. 40).

Example 7 Optimization of Fluorescein Binders

7.1. Construction of L-CDR3 and H-CDR2 Library Cassettes

A L-CDR3 library cassette was prepared from the oligonucleotide templateCDR3L (5′-TGGAAGCTGAAGACGTGGGCGTGTATTATTGCCAGCAG(TR5)(TRI)₄CCG(TRI)-TTTGGCCAGGGTACGAAAGTT-3′) and primer 5′-AACTTTCGTACCCTGGCC-3′ forsynthesis of the complementary strand, where (TRI) was a trinucleotidemixture representing all amino acids except Cys, (TR5) comprised atrinucleotide mixture representing the 5 codons for Ala, Arg, His, Ser,and Tyr.

A H-CDR2 library cassette was prepared from the oligonucleotide templateCDRsH(5′-AGGGTCTCGAGTGGGTGAGC(TRI)ATT(TRI)₂₋₃(6)₂(TRI)ACC(TRI)TATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGAAAAACACCA-3′),and primer 5′-TGGTGTTTTTCGAATTATCA-3′ for synthesis of the complementarystrand, where (TRI) was a trinucleotide mixture representing all aminoacids except Cys, (6) comprised the incorporation of (A/G) (A/C/G) T,resulting in the formation of 6 codons for Ala, Asn, Asp, Gly, Ser, andThr, and the length distribution being obtained by performing onesubstoichiometric coupling of the (TRI) mixture during synthesis,omitting the capping step normally used in DNA synthesis.

DNA synthesis was performed on a 40 nmole scale, oligos were dissolvedin 1E buffer, purified via gel filtration using spin columns (S-200),and the DNA concentration determined by OD measurement at 260 nm (OD1.0=40 μg/ml).

10 nmole of the oligonucleotide templates and 12 nmole of thecorresponding primers were mixed and annealed at 80° C. for 1 min, andslowly cooled down to 37° C. within 20 to 30 min. The fill-in reactionwas performed for 2 h at 37° C. using Klenow polymerase (2.0 μl) and 250nmole of each dNTP. The excess of dNTPs was removed by gel filtrationusing Nick-Spin columns (Pharmacia), and the double-stranded DNAdigested with BbsI/MscI (L-CDR3), or XhoI/SfuI (H-CDR2) over night at37° C. The cassettes were purified via Nick-Spin columns (Pharmacia),the concentration determined by OD measurement, and the cassettesaliquoted (15 pmole) for being stored at −80° C.

7.2 Library Cloning:

DNA was prepared from the collection of FITC binding clones obtained inExample 2 (approx. 10⁴ to clones). The collection of scFv fragments wasisolated via XbaI/EcoRI digest. The vector pCAL4 (100 fmole, 10 μg)described in Example 4.3 was similarly digested with XbaI/EcoRI,gel-purified and ligated with 300 fmole of the scFv fragment collectionover night at 16° C. The ligation mixture was isopropanol precipitated,air-dried, and the pellets were redissolved in 100 μl of dd H₂O. Theligation mixture was mixed with 1 ml of freshly preparedelectrocompetent SCS 101 cells (for optimization of L-CDR3), or XL1 Bluecells (for optimization of H-CDR2) on ice. One round of electroporationwas performed and the transformants were eluted in SOC medium, shaken at37° C. for 30 minutes, and an aliquot plated out on LB plates(Amp/Tet/Glucose) at 37° C. for 6-9 hrs. The number of transformants was5×10⁴.

Vector DNA (100 μg) was isolated and digested (sequence and restrictionmap of sCH3κ2 see FIG. 8) with BbsI/MscI for optimization of L-CDR3, orXhoI/NspV for optimization of H-CDR2. 10 μg of purified vector fragments(5 pmole) were ligated with 15 pmole of the L-CDR3 or H-CDR2 librarycassettes over night at 16° C. The ligation mixtures were isopropanolprecipitated, air-dried, and the pellets were redissolved in 100 μl ofdd H₂O. The ligation mixtures were mixed with 1 ml of freshly preparedelectrocompetent XL1 Blue cells on ice. Electroporation was performedand the transformants were eluted in SOC medium and shaken at 37° C. for30 minutes. An aliquot was plated out on LB plates (Amp/Tet/Glucose) at37° C. for 6-9 hrs. The number of transformants (library size) wasgreater than 10⁸ for both libraries. The libraries were stored asglycerol cultures.

7.3. Biopanning

This was performed as described for the initial H3κ2H-CDR3 library inExample 2.1. Optimized scFvs binding to FITC could be characterized andanalyzed as described in Example 2.2 and 2.3, and further rounds ofoptimization could be made if necessary.

REFERENCES

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Winter, G., Griffiths, A. D., Hawkins, R. E. & Hoogenboom, H. R., Ann.Rev. Immunol. 12, 433-455 (1994). TABLE 1A Human kappa germline genesegments Used Name¹ Reference² Family³ Germline genes⁴ Vk1-1 9 1 O8;O18; DPK1 Vk1-2 1 1 L14; DPK2 Vk1-3 2 1 L15(1); HK101; HK146; HK189Vk1-4 9 1 L11 Vk1-5 2 1 A30 Vk1-6 1 1 LFVK5 Vk1-7 1 1 LFVK431 Vk1-8 1 1L1; HK137 Vk1-9 1 1 A20; DPK4 Vk1-10 1 1 L18; Va″ Vk1-11 1 1 L4; L18;Va′; V4a Vk1-12 2 1 L5; L19(1); Vb; Vb4; DPK5; L19(2); Vb″; DPK6 Vk1-132 1 L15(2); HK134; HK166; DPK7 Vk1-14 8 1 L8; Vd; DPK8 Vk1-15 8 1 L9; VeVk1-16 1 1 L12(1); HK102; V1 Vk1-17 2 1 L12(2) Vk1-18 1 1 O12a (V3b)Vk1-19 6 1 O2; O12; DPK9 Vk1-20 2 1 L24; Ve″; V13; DPK10 Vk1-21 1 1 O4;O14 Vk1-22 2 1 L22 Vk1-23 2 1 L23 Vk2-1 1 2 A2; DPK12 Vk2-2 6 2 O1;O11(1); DPK13 Vk2-3 6 2 O12(2); V3a Vk2-4 2 2 L13 Vk2-5 1 2 DPK14 Vk2-64 2 A3; A19; DPK15 Vk2-7 4 2 A29; DPK27 Vk2-8 4 2 A13 Vk2-9 1 2 A23Vk2-10 4 2 A7; DPK17 Vk2-11 4 2 A17; DPK18 Vk2-12 4 2 A1; DPK19 Vk3-1 113 A11; humkv305; DPK20 Vk3-2 1 3 L20; Vg″ Vk3-3 2 3 L2; L16; humkv328;humkv328h2; humkv328h5; DPK21 Vk3-4 11 3 A27; humkv325; VkRF; DPK22Vk3-5 2 3 L25; DPK23 Vk3-6 2 3 L10(1) Vk3-7 7 3 L10(2) Vk3-8 7 3 L6; VgVk4-1 3 4 B3; VkIV; DPK24 Vk5-1 10 5 B2; EV15 Vk6-1 12 6 A14; DPK25Vk6-2 12 6 A10; A26; DPK26 Vk7-1 5 7 B1

TABLE 1B Human lambda germline gene segments Used Name¹ Reference²Family³ Germline genes⁴ DPL1 1 1 DPL2 1 1 HUMLV1L1 DPL3 1 1 HUMLV122DPL4 1 1 VLAMBDA 1.1 HUMLV117 2 1 DPL5 1 1 HUMLV117D DPL6 1 1 DPL7 1 1IGLV1S2 DPL8 1 1 HUMLV1042 DPL9 1 1 HUMLV101 DPL10 1 2 VLAMBDA 2.1 3 2DPL11 1 2 DPL12 1 2 DPL13 1 2 DPL14 1 2 DPL16 1 3 Humlv418; IGLV3S1DPL23 1 3 VI III.1 Humlv318 4 3 DPL18 1 7 4A; HUMIGLVA DPL19 1 7 DPL21 18 VL8.1 HUMLV801 5 8 DPL22 1 9 DPL24 1 unassigned VLAMBDA N.2 gVLX-4.4 610

TABLE 1C Human heavy chain germline gene segments Used Name¹ Reference²Family³ Germline genes⁴ VH1-12-1 19 1 DP10; DA-2; DA-6 VH1-12-8 22 1RR.VH1.2 VH1-12-2 6 1 hv1263 VH1-12-9 7 1 YAC-7; RR.VH1.1; 1-69 VH1-12-319 1 DP3 VH1-12-4 19 1 DP21; 4d275a; VH7a VH1-12-5 18 1 I-4.1b; V1-4.1bVH1-12-6 21 1 1D37; VH7b; 7-81; YAC-10 VH1-12-7 19 1 DP14; VH1GRR; V1-18VH1-13-1 10 1 71-5; DP2 VH1-13-2 10 1 E3-10 VH1-13-3 19 1 DP1 VH1-13-412 1 V35 VH1-13-5 8 1 V1-2b VH1-13-6 18 1 I-2; DP75 VH1-13-7 21 1 V1-2VH1-13-8 19 1 DP8 VH1-13-9 3 1 1-1 VH1-13-10 19 1 DP12 VH1-13-11 15 1V13C VH1-13-12 18 1 I-3b; DP25; V1-3b VH1-13-13 3 1 1-92 VH1-13-14 18 1I-3; V1-3 VH1-13-15 19 1 DP15; V1-8 VH1-13-16 3 1 21-2; 3-1; DP7; V1-46VH1-13-17 16 1 HG3 VH1-13-18 19 1 DP4; 7-2; V1-45 VH1-13-19 27 1 COS 5VH1-1X-1 19 1 DP5; 1-24P VH2-21-1 18 2 II-5b VH2-31-1 2 2 VH2S12-1VH2-31-2 2 2 VH2S12-7 VH2-31-3 2 2 VH2S12-9; DP27 VH2-31-4 2 2 VH2S12-10VH2-31-5 14 2 V2-26; DP26; 2-26 VH2-31-6 15 2 VF2-26 VH2-31-7 19 2 DP28;DA-7 VH2-31-14 7 2 YAC-3; 2-70 VH2-31-8 2 2 VH2S12-5 VH2-31-9 2 2VH2S12-12 VH2-31-10 18 2 II-5; V2-5 VH2-31-11 2 2 VH2S12-2; VH2S12-8VH2-31-12 2 2 VH2S12-4; VH2S12-6 VH2-31-13 2 2 VH2S12-14 VH3-11-1 13 3v65-2; DP44 VH3-11-2 19 3 DP45 VH3-11-3 3 3 13-2; DP48 VH3-11-4 19 3DP52 VH3-11-5 14 3 v3-13 VH3-11-6 19 3 DP42 VH3-11-7 3 3 8-1B; YAC-5;3-66 VH3-11-8 14 3 V3-53 VH3-13-1 3 3 22-2B; DP35; V3-11 VH3-13-5 19 3DP59; VH19; V3-35 VH3-13-6 25 3 f1-p1; DP61 VH3-13-7 19 3 DP46; GL-SJ2;COS 8; hv3005; hv3005f3; 3d21b; 56p1 VH3-13-8 24 3 VH26 VH3-13-9 5 3vh26c VH3-13-10 19 3 DP47; VH26; 3-23 VH3-13-11 3 3 1-91 VH3-13-12 19 3DP58 VH3-13-13 3 3 1-9III; DP49; 3-30; 3d28.1 VH3-13-14 24 3 3019B9;DP50; 3-33; 3d277 VH3-13-15 27 3 COS 3 VH3-13-16 19 3 DP51 VH3-13-17 163 H11 VH3-13-18 19 3 DP53; COS 6; 3-74; DA-8 VH3-13-19 19 3 DP54;VH3-11; V3-7 VH3-13-20 14 3 V3-64; YAC-6 VH3-13-21 14 3 V3-48 VH3-13-2214 3 V3-43; DP33 VH3-13-23 14 3 V3-33 VH3-13-24 14 3 V3-21; DP77VH3-13-25 14 3 V3-20; DP32 VH3-13-26 14 3 V3-9; DP31 VH3-14-1 3 3 12-2;DP29; 3-72; DA-3 VH3-14-4 7 3 YAC-9; 3-73; MTGL VH3-14-2 4 3 VHD26VH3-14-3 19 3 DP30 VH3-1X-1 1 3 LSG8.1; LSG9.1; LSG10.1; HUM12IGVH;HUM13IGVH VH3-1X-2 1 3 LSG11.1; HUM4IGVH VH3-1X-3 3 3 9-1; DP38; LSG7.1;RCG1.1; LSG1.1; LSG3.1; LSG5.1; HUM15IGVH; HUM2IGVH; HUM9IGVH VH3-1X-4 13 LSG4.1 VH3-1X-5 1 3 LSG2.1 VH3-1X-6 1 3 LSG6.1; HUM10IGVH VH3-1X-7 183 3-15; V3-15 VH3-1X-8 1 3 LSG12.1; HUM5IGVH VH3-1X-9 14 3 V3-49VH4-11-1 22 4 Tou-VH4.21 VH4-11-2 17 4 VH4.21; DP63; VH5; 4d76; V4-34VH4-11-3 23 4 4.44 VH4-11-4 23 4 4.44.3 VH4-11-5 23 4 4.36 VH4-11-6 23 44.37 VH4-11-7 18 4 IV-4; 4.35; V4-4 VH4-11-8 17 4 VH4.11; 3d197d; DP71;58p2 VH4-11-9 20 4 H7 VH4-11-10 20 4 H8 VH4-11-11 20 4 H9 VH4-11-12 17 4VH4.16 VH4-11-13 23 4 4.38 VH4-11-14 17 4 VH4.15 VH4-11-15 11 4 58VH4-11-16 10 4 71-4; V4-59 VH4-21-1 11 4 11 VH4-21-2 17 4 VH4.17;VH4.23; 4d255; 4.40; DP69 VH4-21-3 17 4 VH4.19; 79; V4-4b VH4-21-4 19 4DP70; 4d68; 4.41 VH4-21-5 19 4 DP67; VH4-4B VH4-21-6 17 4 VH4.22; VHSP;VH-JA VH4-21-7 17 4 VH4.13; 1-9II; 12G-1; 3d28d; 4.42; DP68; 4-28VH4-21-8 26 4 hv4005; 3d24d VH4-21-9 17 4 VH4.14 VH4-31-1 23 4 4.34;3d230d; DP78 VH4-31-2 23 4 4.34.2 VH4-31-3 19 4 DP64; 3d216d VH4-31-4 194 DP65; 4-31; 3d277d VH4-31-5 23 4 4.33; 3d75d VH4-31-6 20 4 H10VH4-31-7 20 4 H11 VH4-31-8 23 4 4.31 VH4-31-9 23 4 4.32 VH4-31-10 20 43d277d VH4-31-11 20 4 3d216d VH4-31-12 20 4 3d279d VH4-31-13 17 4VH4.18; 4d154; DP79 VH4-31-14 8 4 V4-39 VH4-31-15 11 4 2-1; DP79VH4-31-16 23 4 4.30 VH4-31-17 17 4 VH4.12 VH4-31-18 10 4 71-2; DP66VH4-31-19 23 4 4.39 VH4-31-20 8 4 V4-61 VH5-12-1 9 5 VH251; DP73; VHVCW;51-R1; VHVLB; VHVCH; VHVTT; VHVAU; VHVBLK; VhAU; V5-51 VH5-12-2 17 5VHVJB VH5-12-3 3 5 1-v; DP80; 5-78 VH5-12-4 9 5 VH32; VHVRG; VHVMW;5-2R1 VH6-35-1 4 6 VHVI; VH6; VHVIIS; VHVITE: VHVIJB; VHVICH; VHVICW;VHVIBLK; VHVIMW; DP74; 6-1G1; V6-1

TABLE 2A rearranged human kappa sequences Computed Germline Diff. to %diff. to Name¹ aa² family³ gene⁴ germline⁵ germline⁶ Reference⁷ III-3R108 1 O8 1 1.1% 70 No. 86 109 1 O8 3 3.2% 80 AU 108 1 O8 6 6.3% 103 ROY108 1 O8 6 6.3% 43 IC4 108 1 O8 6 6.3% 70 HIV-B26 106 1 O8 3 3.2% 8 GRI108 1 O8 8 8.4% 30 AG 106 1 O8 8 8.6% 116 REI 108 1 O8 9 9.5% 86 CLLPATIENT 16 88 1 O8 2 2.3% 122 CLL PATIENT 14 87 1 O8 2 2.3% 122 CLLPATIENT 15 88 1 O8 2 2.3% 122 GM4672 108 1 O8 11 11.6% 24 HUM. YFC51.1108 1 O8 12 12.6% 110 LAY 108 1 O8 12 12.6% 48 HIV-b13 106 1 O8 9 9.7% 8MAL-NaCl 108 1 O8 13 13.7% 102 STRAb SA-1A 108 1 O2 0 0.0% 120 HuVHCAMP108 1 O8 13 13.7% 100 CRO 108 1 O2 10 10.5% 30 Am 107 108 1 O2 12 12.6%108 WALKER 107 1 O2 4 4.2% 57 III-2R 109 1 A20 0 0.0% 70 FOG1-A4 107 1A20 4 4.2% 41 HK137 95 1 L1 0 0.0% 10 CEA4-8A 107 1 O2 7 7.4% 41 Va′ 951 L4 0 0.0% 90 TR1.21 108 1 O2 4 4.2% 92 HAU 108 1 O2 6 6.3% 123 HK10295 1 L12(1) 0 0.0% 9 H20C3K 108 1 L12(2) 3 3.2% 125 CHEB 108 1 O2 7 7.4%5 HK134 95 1 L15(2) 0 0.0% 10 TEL9 108 1 O2 9 9.5% 73 TR1.32 103 1 O2 33.2% 92 RF-KES1 97 1 A20 4 4.2% 121 WES 108 1 L5 10 10.5% 61 DILp1 95 1O4 1 1.1% 70 SA-4B 107 1 L12(2) 8 8.4% 120 HK101 95 1 L15(1) 0 0.0% 9TR1.23 108 1 O2 5 5.3% 92 HF2-1/17 108 1 A30 0 0.0% 4 2E7 108 1 A30 11.1% 62 33.C9 107 1 L12(2) 7 7.4% 126 3D6 105 1 L12(2) 2 2.1% 34 I-2a108 1 L8 8 8.4% 70 RF-KL1 97 1 L8 4 4.2% 121 TNF-E7 108 1 A30 9 9.5% 41TR1.22 108 1 O2 7 7.4% 92 HIV-B35 106 1 O2 2 2.2% 8 HIV-b22 106 1 O2 22.2% 8 HIV-b27 106 1 O2 2 2.2% 8 HIV-B8 107 1 O2 10 10.8% 8 HIV-b3 107 1O2 10 10.8% 8 RF-SJ5 95 1 A30 5 5.3% 113 GAL(I) 108 1 A30 6 6.3% 64R3.5H5G 108 1 O2 6 6.3% 70 HIV-b14 106 1 A20 2 2.2% 8 TNF-E1 105 1 L5 88.4% 41 WEA 108 1 A30 8 8.4% 37 EU 108 1 L12(2) 5 5.3% 40 FOG1-G8 108 1L8 11 11.6% 41 1X7RG1 108 1 L1 8 8.4% 70 BLI 108 1 L8 3 3.2% 72 KUE 1081 L12(2) 11 11.6% 32 LUNm01 108 1 L12(2) 10 10.5% 6 HIV-b1 106 1 A20 44.3% 8 HIV-s4 103 1 O2 2 2.2% 8 CAR 107 1 L12(2) 11 11.7% 79 BR 107 1L12(2) 11 11.6% 50 CLL PATIENT 10 88 1 O2 0 0.0% 122 CLL PATIENT 12 88 1O2 0 0.0% 122 KING 108 1 L12(2) 12 12.6% 30 V13 95 1 L24 0 0.0% 46 CLLPATIENT 11 87 1 O2 0 0.0% 122 CLL PATIENT 13 87 1 O2 0 0.0% 122 CLLPATIENT 9 88 1 O12 1 1.1% 122 HIV-B2 106 1 A20 9 9.7% 8 HIV-b2 106 1 A209 9.7% 8 CLL PATIENT 5 88 1 A20 1 1.1% 122 CLL PATIENT 1 88 1 L8 2 2.3%122 CLL PATIENT 2 88 1 L8 0 0.0% 122 CLL PATIENT 7 88 1 L5 0 0.0% 122CLL PATIENT 8 88 1 L5 0 0.0% 122 HIV-b5 105 1 L5 11 12.0% 8 CLL PATIENT3 87 1 L8 1 1.1% 122 CLL PATIENT 4 88 1 L9 0 0.0% 122 CLL PATIENT 18 851 L9 6 7.1% 122 CLL PATIENT 17 86 1 L12(2) 7 8.1% 122 HIV-b20 107 3 A2711 11.7% 8 2C12 108 1 L12(2) 20 21.1% 68 1B11 108 1 L12(2) 20 21.1% 681H1 108 1 L12(2) 21 22.1% 68 2A12 108 1 L12(2) 21 22.1% 68 CUR 109 3 A270 0.0% 66 GLO 109 3 A27 0 0.0% 16 RF-TS1 96 3 A27 0 0.0% 121 GAR′ 109 3A27 0 0.0% 67 FLO 109 3 A27 0 0.0% 66 PIE 109 3 A27 0 0.0% 91 HAH 14.1109 3 A27 1 1.0% 51 HAH 14.2 109 3 A27 1 1.0% 51 HAH 16.1 109 3 A27 11.0% 51 NOV 109 3 A27 1 1.0% 52 33.F12 108 3 A27 1 1.0% 126 8E10 110 3A27 1 1.0% 25 TH3 109 3 A27 1 1.0% 25 HIC (R) 108 3 A27 0 0.0% 51 SON110 3 A27 1 1.0% 67 PAY 109 3 A27 1 1.0% 66 GOT 109 3 A27 1 1.0% 67mAbA6H4C5 109 3 A27 1 1.0% 12 BOR′ 109 3 A27 2 2.1% 84 RF-SJ3 96 3 A27 22.1% 121 SIE 109 3 A27 2 2.1% 15 ESC 109 3 A27 2 2.1% 98 HEW′ 110 3 A272 2.1% 98 YES8c 109 3 A27 3 3.1% 33 TI 109 3 A27 3 3.1% 114 mAb113 109 3A27 3 3.1% 71 HEW 107 3 A27 0 0.0% 94 BRO 106 3 A27 0 0.0% 94 ROB 106 3A27 0 0.0% 94 NG9 96 3 A27 4 4.2% 11 NEU 109 3 A27 4 4.2% 66 WOL 109 3A27 4 4.2% 2 35G6 109 3 A27 4 4.2% 59 RF-SJ4 109 3 A11 0 0.0% 88 KAS 1093 A27 4 4.2% 84 BRA 106 3 A27 1 1.1% 94 HAH 106 3 A27 1 1.1% 94 HIC 1053 A27 0 0.0% 94 FS-2 109 3 A27 6 6.3% 87 JH′ 107 3 A27 6 6.3% 38 EV1-15109 3 A27 6 6.3% 83 SCA 108 3 A27 6 6.3% 65 mAb112 109 3 A27 6 6.3% 71SIC 103 3 A27 3 3.3% 94 SA-4A 109 3 A27 6 6.3% 120 SER 108 3 A27 6 6.3%98 GOL′ 109 3 A27 7 7.3% 82 B5G10K 105 3 A27 9 9.7% 125 HG2B10K 110 3A27 9 9.4% 125 Taykv322 105 3 A27 5 5.4% 52 CLL PATIENT 24 89 3 A27 11.1% 122 HIV-b24 107 3 A27 7 7.4% 8 HIV-b6 107 3 A27 7 7.4% 8 Taykv31099 3 A27 1 1.1% 52 KA3D1 108 3 L6 0 0.0% 85 19.E7 107 3 L6 0 0.0% 126rsv6L 109 3 A27 12 12.5% 7 Taykv320 98 3 A27 1 1.2% 52 Vh 96 3 L10(2) 00.0% 89 LS8 108 3 L6 1 1.1% 109 LS1 108 3 L6 1 1.1% 109 LS2S3-3 107 3 L62 2.1% 99 LS2 108 3 L6 1 1.1% 109 LS7 108 3 L6 1 1.1% 109 LS2S3-4d 107 3L6 2 2.1% 99 LS2S3-4a 107 3 L6 2 2.1% 99 LS4 108 3 L6 1 1.1% 109 LS6 1083 L6 1 1.1% 109 LS2S3-10a 107 3 L6 2 2.1% 99 LS2S3-8c 107 3 L6 2 2.1% 99LS5 108 3 L6 1 1.1% 109 LS2S3-5 107 3 L6 3 3.2% 99 LUNm03 109 3 A27 1313.5% 6 IARC/BL41 108 3 A27 13 13.7% 55 slkv22 99 3 A27 3 3.5% 13 POP108 3 L6 4 4.2% 111 LS2S3-10b 107 3 L6 3 3.2% 99 LS2S3-8f 107 3 L6 33.2% 99 LS2S3-12 107 3 L6 3 3.2% 99 HIV-B30 107 3 A27 11 11.7% 8 HIV-B20107 3 A27 11 11.7% 8 HIV-b3 108 3 A27 11 11.7% 8 HIV-s6 104 3 A27 9 9.9%8 YSE 107 3 L2/L16 1 1.1% 72 POM 109 3 L2/L16 9 9.4% 53 Humkv328 95 3L2/L16 1 1.1% 19 CLL 109 3 L2/L16 3 3.2% 47 LES 96 3 L2/L16 3 3.2% 38HIV-s5 104 3 A27 11 12.1% 8 HIV-s7 104 3 A27 11 12.1% 8 slkv1 99 3 A27 78.1% 13 Humka31es 95 3 L2/L16 4 4.2% 18 slkv12 101 3 A27 8 9.2% 13RF-TS2 95 3 L2/L16 3 3.2% 121 II-1 109 3 L2/L16 4 4.2% 70 HIV-s3 105 3A27 13 14.3% 8 RF-TMC1 96 3 L6 10 10.5% 121 GER 109 3 L2/L16 7 7.4% 75GF4/1.1 109 3 L2/L16 8 8.4% 36 mAb114 109 3 L2/L16 6 6.3% 71 HIV-loop13109 3 L2/L16 7 7.4% 8 bkv16 86 3 L6 1 1.2% 13 CLL PATIENT 29 86 3 L6 11.2% 122 slkv9 98 3 L6 3 3.5% 13 bkv17 99 3 L6 1 1.2% 13 slkv14 99 3 L61 1.2% 13 slkv16 101 3 L6 2 2.3% 13 bkv33 101 3 L6 4 4.7% 13 slkv15 99 3L6 2 2.3% 13 bkv6 100 3 L6 3 3.5% 13 R6B8K 108 3 L2/L16 12 12.6% 125 AL700 107 3 L2/L16 9 9.5% 117 slkv11 100 3 L2/L16 3 3.5% 13 slkv4 97 3 L64 4.8% 13 CLL PATIENT 26 87 3 L2/L16 1 1.1% 122 AL Se124 103 3 L2/L16 99.5% 117 slkv13 100 3 L2/L16 6 7.0% 13 bkv7 100 3 L2/L16 5 5.8% 13 bkv22100 3 L2/L16 6 7.0% 13 CLL PATIENT 27 84 3 L2/L16 0 0.0% 122 bkv35 100 3L6 8 9.3% 13 CLL PATIENT 25 87 3 L2/L16 4 4.6% 122 slkv3 86 3 L2/L16 78.1% 13 slkv7 99 1 O2 7 8.1% 13 HuFd79 111 3 L2/L16 24 24.2% 21 RAD 99 3A27 9 10.3% 78 CLL PATIENT 28 83 3 L2/L16 4 4.8% 122 REE 104 3 L2/L16 2527.2% 95 FR4 99 3 A27 8 9.2% 77 MD3.3 92 3 L6 1 1.3% 54 MD3.1 92 3 L6 00.0% 54 GA3.6 92 3 L6 2 2.6% 54 M3.5N 92 3 L6 3 3.8% 54 WEI′ 82 3 A27 00.0% 65 MD3.4 92 3 L2/L16 1 1.3% 54 MD3.2 91 3 L6 3 3.8% 54 VER 97 3 A2719 22.4% 20 CLL PATIENT 30 78 3 L6 3 3.8% 122 M3.1N 92 3 L2/L16 1 1.3%54 MD3.6 91 3 L2/L16 0 0.0% 54 MD3.8 91 3 L2/L16 0 0.0% 54 GA3.4 92 3 L67 9.0% 54 M3.6N 92 3 A27 0 0.0% 54 MD3.10 92 3 A27 0 0.0% 54 MD3.13 91 3A27 0 0.0% 54 MD3.7 93 3 A27 0 0.0% 54 MD3.9 93 3 A27 0 0.0% 54 GA3.1 933 A27 6 7.6% 54 bkv32 101 3 A27 5 5.7% 13 GA3.5 93 3 A27 5 6.3% 54 GA3.792 3 A27 7 8.9% 54 MD3.12 92 3 A27 2 2.5% 54 M3.2N 90 3 L6 6 7.8% 54MD3.5 92 3 A27 1 1.3% 54 M3.4N 91 3 L2/L16 8 10.3% 54 M3.8N 91 3 L2/L167 9.0% 54 M3.7N 92 3 A27 3 3.8% 54 GA3.2 92 3 A27 9 11.4% 54 GA3.8 93 3A27 4 5.1% 54 GA3.3 92 3 A27 8 10.1% 54 M3.3N 92 3 A27 5 6.3% 54 B6 83 3A27 8 11.3% 78 E29.1 KAPPA 78 3 L2/L16 0 0.0% 22 SCW 108 1 O8 12 12.6%31 REI-based CAMPATH-9 107 1 O8 14 14.7% 39 RZ 107 1 O8 14 14.7% 50 BI108 1 O8 14 14.7% 14 AND 107 1 O2 13 13.7% 69 2A4 109 1 O2 12 12.6% 23KA 108 1 O8 19 20.0% 107 MEV 109 1 O2 14 14.7% 29 DEE 106 1 O2 13 14.0%76 OU(IOC) 108 1 O2 18 18.9% 60 HuRSV19VK 111 1 O8 21 21.0% 115 SP2 1081 O2 17 17.9% 93 BJ26 99 1 O8 21 24.1% 1 NI 112 1 O8 24 24.2% 106 BMA0310EUCIV2 106 1 L12(1) 21 22.3% 105 CLL PATIENT 6 71 1 A20 0 0.0% 122BJ19 85 1 O8 16 21.9% 1 GM 607 113 2 A3 0 0.0% 58 R5A3K 114 2 A3 1 1.0%125 R1C8K 114 2 A3 1 1.0% 125 VK2.R149 113 2 A3 2 2.0% 118 TR1.6 109 2A3 4 4.0% 92 TR1.37 104 2 A3 5 5.0% 92 FS-1 113 2 A3 6 6.0% 87 TR1.8 1102 A3 6 6.0% 92 NIM 113 2 A3 8 8.0% 28 Inc 112 2 A3 11 11.0% 35 TEW 107 2A3 6 6.4% 96 CUM 114 2 O1 7 6.9% 44 HRF1 71 2 A3 4 5.6% 124 CLL PATIENT19 87 2 A3 0 0.0% 122 CLL PATIENT 20 87 2 A3 0 0.0% 122 MIL 112 2 A3 1616.2% 26 FR 113 2 A3 20 20.0% 101 MAL-Urine 83 1 O2 6 8.6% 102 Taykv30673 3 A27 1 1.6% 52 Taykv312 75 3 A27 1 1.6% 52 HIV-b29 93 3 A27 14 17.5%8 1-185-37 110 3 A27 0 0.0% 119 1-187-29 110 3 A27 0 0.0% 119 TT117 1103 A27 9 9.4% 63 HIV-loop8 108 3 A27 16 16.8% 8 rsv23L 108 3 A27 16 16.8%7 HIV-b7 107 3 A27 14 14.9% 8 HIV-b11 107 3 A27 15 16.0% 8 HIV-LC1 107 3A27 19 20.2% 8 HIV-LC7 107 3 A27 20 21.3% 8 HIV-LC22 107 3 A27 21 22.3%8 HIV-LC13 107 3 A27 21 22.3% 8 HIV-LC3 107 3 A27 21 22.3% 8 HIV-LC5 1073 A27 21 22.3% 8 HIV-LC28 107 3 A27 21 22.3% 8 HIV-b4 107 3 A27 22 23.4%8 CLL PATIENT 31 87 3 A27 15 17.2% 122 HIV-loop2 108 3 L2/L16 17 17.9% 8HIV-loop35 108 3 L2/L16 17 17.9% 8 HIV-LC11 107 3 A27 23 24.5% 8HIV-LC24 107 3 A27 23 24.5% 8 HIV-b12 107 3 A27 24 25.5% 8 HIV-LC25 1073 A27 24 25.5% 8 HIV-b21 107 3 A27 24 25.5% 8 HIV-LC26 107 3 A27 2627.7% 8 G3D10K 108 1 L12(2) 12 12.6% 125 TT125 108 1 L5 8 8.4% 63 HIV-s2103 3 A27 28 31.1% 8 265-695 108 1 L5 7 7.4% 3 2-115-19 108 1 A30 2 2.1%119 rsv13L 107 1 O2 20 21.1% 7 HIV-b18 106 1 O2 14 15.1% 8 RF-KL5 98 3L6 36 36.7% 97 ZM1-1 113 2 A17 7 7.0% 3 HIV-s8 103 1 O8 16 17.8% 8K-EV15 95 5 B2 0 0.0% 112 RF-TS3 100 2 A23 0 0.0% 121 HF-21/28 111 2 A171 1.0% 17 RPMI6410 113 2 A17 1 1.0% 42 JC11 113 2 A17 1 1.0% 49 O-81 1142 A17 5 5.0% 45 FK-001 113 4 B3 0 0.0% 81 CD5+.28 101 4 83 1 1.0% 27 LEN114 4 B3 1 1.0% 104 UC 114 4 B3 1 1.0% 111 CD5+.5 101 4 B3 1 1.0% 27CD5+.26 101 4 B3 1 1.0% 27 CD5+.12 101 4 B3 2 2.0% 27 CD5+.23 101 4 B3 22.0% 27 CD5+.7 101 4 B3 2 2.0% 27 VJI 113 4 B3 3 3.0% 56 LOC 113 4 B3 33.0% 72 MAL 113 4 B3 3 3.0% 72 CD5+.6 101 4 B3 3 3.0% 27 H2F 113 4 B3 33.0% 70 PB17IV 114 4 B3 4 4.0% 74 CD5+.27 101 4 B3 4 4.0% 27 CD5+.9 1014 B3 4 4.0% 27 CD5−.28 101 4 B3 5 5.0% 27 CD5−.26 101 4 B3 6 5.9% 27CD5+.24 101 4 B3 6 5.9% 27 CD5+.10 101 4 B3 6 5.9% 27 CD5−.19 101 4 B3 65.9% 27 CD5−.18 101 4 B3 7 6.9% 27 CD5−.16 101 4 B3 8 7.9% 27 CD5−.24101 4 B3 8 7.9% 27 CD5−.17 101 4 B3 10 9.9% 27 MD4.1 92 4 B3 0 0.0% 54MD4.4 92 4 B3 0 0.0% 54 MD4.5 92 4 B3 0 0.0% 54 MD4.6 92 4 B3 0 0.0% 54MD4.7 92 4 B3 0 0.0% 54 MD4.2 92 4 B3 1 1.3% 54 MD4.3 92 4 B3 5 6.3% 54CLL PATIENT 22 87 2 A17 2 2.3% 122 CLL PATIENT 23 84 2 A17 2 2.4% 122

TABLE 2B rearranged human lambda sequences Computed Germline Diff. to %diff. to Name¹ aa² family³ gene⁴ germline⁵ germline⁶ Reference⁷ WAH 1101 DPL3 7 7% 68 1B9/F2 112 1 DPL3 7 7% 9 DIA 112 1 DPL2 7 7% 36 mAb67 891 DPL3 0 0% 29 HiH2 110 1 DPL3 12 11% 3 NIG-77 112 1 DPL2 9 9% 72 OKA112 1 DPL2 7 7% 84 KOL 112 1 DPL2 12 11% 40 T2:C5 111 1 DPL5 0 0% 6T2:C14 110 1 DPL5 0 0% 6 PR-TS1 110 1 DPL5 0 0% 55 4G12 111 1 DPL5 1 1%35 KIM46L 112 1 HUMLV117 0 0% 8 Fog-B 111 1 DPL5 3 3% 31 9F2L 111 1 DPL53 3% 79 mAb111 110 1 DPL5 3 3% 48 PHOX15 111 1 DPL5 4 4% 49 BL2 111 1DPL5 4 4% 74 NIG-64 111 1 DPL5 4 4% 72 RF-SJ2 100 1 DPL5 6 6% 78 AL EZI112 1 DPL5 7 7% 41 ZIM 112 1 HUMLV117 7 7% 18 RF-SJ1 100 1 DPL5 9 9% 78IGLV1.1 98 1 DPL4 0 0% 1 NEW 112 1 HUMLV117 11 10% 42 CB-201 87 1 DPL2 11% 62 MEM 109 1 DPL2 6 6% 50 H210 111 2 DPL10 4 4% 45 NOV 110 2 DPL10 88% 25 NEI 111 2 DPL10 8 8% 24 AL MC 110 2 DPL11 6 6% 28 MES 112 2 DPL118 8% 84 FOG1-A3 111 2 DPL11 9 9% 27 AL NOV 112 2 DPL11 7 7% 28 HMST-1110 2 DPL11 4 4% 82 HBW4-1 108 2 DPL12 9 9% 52 WH 110 2 OPL11 11 11% 3411-50 110 2 DPL11 7 7% 82 HBp2 110 2 DPL12 8 8% 3 NIG-84 113 2 DPL11 1211% 73 VIL 112 2 DPL11 9 9% 58 TRO 111 2 DPL12 10 10% 61 ES492 108 2DPL11 15 15% 76 mAb216 89 2 DPL12 1 1% 7 BSA3 109 3 DPL16 0 0% 49 THY-29110 3 DPL16 0 0% 27 PR-TS2 108 3 DPL16 0 0% 55 E29.1 LAMBDA 107 3 DPL161 1% 13 mAb63 109 3 DPL16 2 2% 29 TEL14 110 3 DPL16 6 6% 49 6H-3C4 108 3DPL16 7 7% 39 SH 109 3 DPL16 7 7% 70 AL GIL 109 3 DPL16 8 8% 23 H6-3C4108 3 DPL16 8 8% 83 V-lambda-2.DS 111 2 DPL11 3 3% 15 8.12 ID 110 2DPL11 3 3% 81 DSC 111 2 DPL11 3 3% 56 PV11 110 2 DPL11 1 1% 56 33.H11110 2 DPL11 4 4% 81 AS17 111 2 DPL11 7 7% 56 SD6 110 2 DPL11 7 7% 56 KS3110 2 DPL11 9 9% 56 PV6 110 2 DPL12 5 5% 56 NGD9 110 2 DPL11 7 7% 56MUC1-1 111 2 DPL11 11 10% 27 A30c 111 2 DPL10 6 6% 56 KS6 110 2 DPL12 66% 56 TEL13 111 2 DPL11 11 10% 49 AS7 110 2 DPL12 6 6% 56 MCG 112 2DPL12 12 11% 20 U266L 110 2 DPL12 13 12% 77 PR-SJ2 110 2 DPL12 14 13% 55BOH 112 2 DPL12 11 10% 37 TOG 111 2 DPL11 19 18% 53 TEL16 111 2 DPL11 1918% 49 No. 13 110 2 DPL10 14 13% 52 BO 112 2 DPL12 18 17% 80 WIN 112 2DPL12 17 16% 11 BUR 104 2 DPL12 15 15% 46 NIG-58 110 2 DPL12 20 19% 69WEIR 112 2 DPL11 26 25% 21 THY-32 111 1 DPL8 8 8% 27 TNF-H9G1 111 1 DPL89 9% 27 mAb61 111 1 DPL3 1 1% 29 LV1L1 98 1 DPL2 0 0% 54 HA 113 1 DPL314 13% 63 LA1L1 111 1 DPL2 3 3% 54 RHE 112 1 DPL1 17 16% 22 K1B12L 113 1DPL8 17 16% 79 LOC 113 1 DPL2 15 14% 84 NIG-51 112 1 DPL2 12 11% 67 NEWM104 1 DPL8 23 22% 10 MD3-4 106 3 DPL23 14 13% 4 COX 112 1 DPL2 13 12% 84HiH10 106 3 DPL23 13 12% 3 VOR 112 1 DPL2 16 15% 16 AL POL 113 1 DPL2 1615% 57 CD4-74 111 1 DPL2 19 18% 27 AMYLOID MOL 102 3 DPL23 15 15% 30OST577 108 3 Humlv318 10 10% 4 NIG-48 113 1 DPL3 42 40% 66 CARR 108 3DPL23 18 17% 19 mAb60 108 3 DPL23 14 13% 29 NIG-68 99 3 DPL23 25 26% 32KERN 107 3 DPL23 26 25% 59 ANT 106 3 DPL23 17 16% 19 LEE 110 3 DPL23 1817% 85 CLE 94 3 DPL23 17 17% 19 VL8 98 8 DPL21 0 0% 81 MOT 110 3Humlv318 23 22% 38 GAR 108 3 DPL23 26 25% 33 32.B9 98 8 DPL21 5 5% 81PUG 108 3 Humlv318 24 23% 19 T1 115 8 HUMLV801 52 50% 6 RF-TS7 96 7DPL18 4 4% 60 YM-1 116 8 HUMLV801 51 49% 75 K6H6 112 8 HUMLV801 20 19%44 K5C7 112 8 HUMLV801 20 19% 44 K5B8 112 8 HUMLV801 20 19% 44 K5G5 1128 HUMLV801 20 19% 44 K4B8 112 8 HUMLV801 19 18% 44 K6F5 112 8 HUMLV80117 16% 44 HIL 108 3 DPL23 22 21% 47 KIR 109 3 DPL23 20 19% 19 CAP 109 3DPL23 19 18% 84 1B8 110 3 DPL23 22 21% 43 SHO 108 3 DPL23 19 18% 19 HAN108 3 DPL23 20 19% 19 cML23 96 3 DPL23 3 3% 12 PR-SJ1 96 3 DPL23 7 7% 55BAU 107 3 DPL23 9 9% 5 TEX 99 3 DPL23 8 8% 19 X(PET) 107 3 DPL23 9 9% 51DOY 106 3 DPL23 9 9% 19 COT 106 3 DPL23 13 12% 19 Pag-1 111 3 Humlv318 550% 31 DIS 107 3 Humlv318 2 2% 19 WIT 108 3 Humlv318 7 7% 19 I.RH 108 3Humlv318 12 11% 19 s1-1 108 3 Humlv318 12 11% 52 DEL 108 3 Humlv318 1413% 17 TYR 108 3 Humlv318 11 10% 19 J.RH 109 3 Humlv318 13 12% 19 THO112 2 DPL13 38 36% 26 LBV 113 1 DPL3 38 36% 2 WLT 112 1 DPL3 33 31% 14SUT 112 2 DPL12 37 35% 65

TABLE 2C rearranged human heavy chain sequences Computed Germline Diff.to % diff. to Name¹ aa² family³ gene⁴ germline⁵ germline⁶ Reference⁷21/28 119 1 VH1-13-12 0 0.0% 31 8E10 123 1 VH1-13-12 0 0.0% 31 MUC1-1118 1 VH1-13-6 4 4.1% 42 gF1 98 1 VH1-13-12 10 10.2% 75 VHGL 1.2 98 1VH1-13-6 2 2.0% 26 HV1L1 98 1 VH1-13-6 0 0.0% 81 RF-TS7 104 1 VH1-13-6 33.1% 96 E55 1.A15 106 1 VH1-13-15 1 1.0% 26 HA1L1 126 1 VH1-13-6 7 7.1%81 UC 123 1 VH1-13-6 5 5.1%

5 WIL2 123 1 VH1-13-6 6 6.1% 55 R3.5H5G 122 1 VH1-13-6 10 10.2% 70 N89P2123 1 VH1-13-16 11 11.2% 77 mAb113 126 1 VH1-13-6 10 10.2% 71 LS2S3-3125 1 VH1-12-7 5 5.1% 98 LS2S3-12a 125 1 VH1-12-7 5 5.1% 98 LS2S3-5 1251 VH1-12-7 5 5.1% 98 LS2S3-12e 125 1 VH1-12-7 5 5.1% 98 LS2S3-4 125 1VH1-12-7 5 5.1% 98 LS2S3-10 125 1 VH1-12-7 5 5.1% 98 LS2S3-12d 125 1VH1-12-7 6 6.1% 98 LS2S3-8 125 1 VH1-12-7 5 5.1% 98 LS2 125 1 VH1-12-7 66.1% 113 LS4 105 1 VH1-12-7 6 6.1% 113 LS5 125 1 VH1-12-7 6 6.1% 113 LS1125 1 VH1-12-7 6 6.1% 113 LS6 125 1 VH1-12-7 6 6.1% 113 LS8 125 1VH1-12-7 7 7.1% 113 THY-29 122 1 VH1-12-7 0 0.0% 42 1B9/F2 122 1VH1-12-7 10 10.2% 21 51P1 122 1 VH1-12-1 0 0.0% 105 NEI 127 1 VH1-12-1 00.0% 55 AND 127 1 VH1-12-1 0 0.0% 55 L7 127 1 VH1-12-1 0 0.0% 54 L22 1241 VH1-12-1 0 0.0% 54 L24 127 1 VH1-12-1 0 0.0% 54 L26 116 1 VH1-12-1 00.0% 54 L33 119 1 VH1-12-1 0 0.0% 54 L34 117 1 VH1-12-1 0 0.0% 54 L36118 1 VH1-12-1 0 0.0% 54 L39 120 1 VH1-12-1 0 0.0% 54 L41 120 1 VH1-12-10 0.0% 54 L42 125 1 VH1-12-1 0 0.0% 54 VHGL 1.8 101 1 VH1-12-1 0 0.0% 26783c 127 1 VH1-12-1 0 0.0% 22 X17115 127 1 VH1-12-1 0 0.0% 37 L25 124 1VH1-12-1 0 0.0% 54 L17 120 1 VH1-12-1 1 1.0% 54 L30 127 1 VH1-12-1 11.0% 54 L37 120 1 VH1-12-1 1 1.0% 54 TNF-E7 116 1 VH1-12-1 2 2.0% 42mAb111 122 1 VH1-12-1 7 7.1% 71 III-2R 122 1 VH1-12-9 3 3.1% 70 KAS 1211 VH1-12-1 7 7.1% 79 YES8c 122 1 VH1-12-1 8 8.2% 34 RF-TS1 123 1VH1-12-1 8 8.2% 82 BOR′ 121 1 VH1-12-8 7 7.1% 79 VHGL 1.9 101 1 VH1-12-18 8.2% 26 mAb410.30F305 117 1 VH1-12-9 5 5.1% 52 EV1-15 127 1 VH1-12-810 10.2% 78 mAb112 122 1 VH1-12-1 11 11.2% 71 EU 117 1 VH1-12-1 11 11.2%28 H210 127 1 VH1-12-1 12 12.2% 66 TRANSGENE 104 1 VH1-12-1 0 0.0% 111CLL2-1 93 1 VH1-12-1 0 0.0% 30 CLL10 13-3 97 1 VH1-12-1 0 0.0% 29 LS7 991 VH1-12-7 4 4.1% 113 ALL7-1 87 1 VH1-12-7 0 0.0% 30 CLL3-1 91 1VH1-12-7 1 1.0% 30 ALL56-1 85 1 VH1-13-8 0 0.0% 30 ALL1-1 87 1 VH1-13-61 1.0% 30 ALL4-1 94 1 VH1-13-8 0 0.0% 30 ALL56 15-4 85 1 VH1-13-8 5 5.1%29 CLL4-1 88 1 VH1-13-1 1 1.0% 30 Au92.1 98 1 VH1-12-5 0 0.0% 49 RF-TS3120 1 VH1-12-5 1 1.0% 82 Au4.1 98 1 VH1-12-5 1 1.0% 49 HP1 121 1VH1-13-6 13 13.3% 110 BLI 127 1 VH1-13-15 5 5.1% 72 No. 13 127 1VH1-12-2 19 19.4% 76 TR1.23 122 1 VH1-13-2 23 23.5% 88 S1-1 125 1VH1-12-2 18 18.4% 76 TR1.10 119 1 VH1-13-12 14 14.3% 88 E55 1.A2 102 1VH1-13-15 3 3.1% 26 SP2 119 1 VH1-13-6 15 15.3% 89 TNF-H9G1 111 1VH1-13-18 2 2.0% 42 G3D10H 127 1 VH1-13-16 19 19.4% 127 TR1.9 118 1VH1-13-12 14 14.3% 88 TR1.8 121 1 VH1-12-1 24 24.5% 88 LUNm01 127 1VH1-13-6 22 22.4% 9 K1B12H 127 1 VH1-12-7 23 23.5% 127 L3B2 99 1VH1-13-6 2 2.0% 46 ss2 100 1 VH1-13-6 2 2.0% 46 No. 86 124 1 VH1-12-1 2020.4% 76 TR1.6 124 1 VH1-12-1 19 19.4% 88 ss7 99 1 VH1-12-7 3 3.1% 46s5B7 102 1 VH1-12-1 0 0.0% 46 s6A3 97 1 VH1-12-1 0 0.0% 46 ss6 99 1VH1-12-1 0 0.0% 46 L2H7 103 1 VH1-13-12 0 0.0% 46 s6BG8 93 1 VH1-13-12 00.0% 46 s6C9 107 1 VH1-13-12 0 0.0% 46 HIV-b4 124 1 VH1-13-12 21 21.4%12 HIV-b12 124 1 VH1-13-12 21 21.4% 12 L3G5 98 1 VH1-13-6 1 1.0% 46 22115 1 VH1-13-6 11 11.2% 118 L2A12 99 1 VH1-13-15 3 3.1% 46 PHOX15 124 1VH1-12-7 20 20.4% 73 LUNm03 127 1 VH1-1X-1 18 18.4% 9 CEA4-8A 129 1VH1-12-7 1 1.0% 42 M60 121 2 VH2-31-3 3 3.0% 103 HiH10 127 2 VH2-31-5 99.0% 4 COR 119 2 VH2-31-2 11 11.0% 91 2-115-19 124 2 VH2-31-11 8 8.1%124 OU 125 2 VH2-31-14 20 25.6% 92 HE 120 2 VH2-31-13 19 19.0% 27 CLL3340-1 78 2 VH2-31-5 2 2.0% 29 E55 3.9 88 3 VH3-11-5 7 7.2% 26 MTFC3 125 3VH3-14-4 21 21.0% 131 MTFC11 125 3 VH3-14-4 21 21.0% 131 MTFJ1 114 3VH3-14-4 21 21.0% 131 MTFJ2 114 3 VH3-14-4 21 21.0% 131 MTFUJ4 100 3VH3-14-4 21 21.0% 131 MTFUJ5 100 3 VH3-14-4 21 21.0% 131 MTFUJ2 100 3VH3-14-4 22 22.0% 131 MTFC8 125 3 VH3-14-4 23 23.0% 131 TD e Vq 113 3VH3-14-4 0 0.0% 16 rMTF 114 3 VH3-14-4 5 5.0% 131 MTFUJ6 100 3 VH3-14-410 10.0% 131 RF-KES 107 3 VH3-14-4 9 9.0% 85 N51P8 126 3 VH3-14-1 9 9.0%77 TEI 119 3 VH3-13-8 21 21.4% 20 33.H11 115 3 VH3-13-19 10 10.2% 129SB1/D8 101 3 VH3-IX-8 14 14.0% 2 38P1 119 3 VH3-11-3 0 0.0% 104 BRO'IGM119 3 VH3-11-3 13 13.4% 19 NIE 119 3 VH3-13-7 15 15.3% 87 3D6 126 3VH3-13-26 5 5.1% 35 ZM1-1 112 3 VH3-11-3 8 8.2% S E55 3.15 110 3VH3-13-26 0 0.0% 26 gF9 108 3 VH3-13-8 15 15.3% 75 THY-32 120 3VH3-13-26 3 3.1% 42 RF-KL5 100 3 VH3-13-26 5 5.1% 96 OST577 122 3VH3-13-13 6 6.1% 5 BO 113 3 VH3-13-19 15 15.3% 10 TT125 121 3 VH3-13-1015 15.3% 64 2-115-58 127 3 VH3-13-10 11 11.2% 124 KOL 126 3 VH3-13-14 1616.3% 102 mAb60 118 3 VH3-13-17 14 14.3% 45 RF-AN 106 3 VH3-13-26 8 8.2%85 BUT 115 3 VH3-11-6 13 13.4% 119 KOL-based CAMPATH-9 118 3 VH3-13-1316 16.3% 41 B1 119 3 VH3-13-19 13 13.3% 53 N98P1 127 3 VH3-13-1 13 13.3%77 TT117 107 3 VH3-13-10 12 12.2% 64 WEA 114 3 VH3-13-12 15 15.3% 40 HIL120 3 VH3-13-14 14 14.3% 23 s5A10 97 3 VH3-13-14 0 0.0% 46 s5D11 98 3VH3-13-7 0 0.0% 46 s6C8 100 3 VH3-13-7 0 0.0% 46 s6H12 98 3 VH3-13-7 00.0% 46 VH10.7 119 3 VH3-13-14 16 16.3% 128 HIV-loop2 126 3 VH3-13-7 1616.3% 12 HIV-loop35 126 3 VH3-13-7 16 16.3% 12 TRO 122 3 VH3-13-1 1313.3% 61 SA-4B 123 3 VH3-13-1 15 15.3% 125 L2B5 98 3 VH3-13-13 0 0.0% 46s6E11 95 3 VH3-13-13 0 0.0% 46 s6H7 100 3 VH3-13-13 0 0.0% 46 ss1 102 3VH3-13-13 0 0.0% 46 ss8 94 3 VH3-13-13 0 0.0% 46 DOB 120 3 VH3-13-26 2121.4% 116 THY-33 115 3 VH3-13-15 20 20.4% 42 NOV 118 3 VH3-13-19 1414.3% 38 rsv13H 120 3 VH3-13-24 20 20.4% 11 L3G11 98 3 VH3-13-20 2 2.0%46 L2E8 99 3 VH3-13-19 0 0.0% 46 L2D10 101 3 VH3-13-10 1 1.0% 46 L2E7 983 VH3-13-10 1 1.0% 46 L3A10 100 3 VH3-13-24 0 0.0% 46 L2E5 97 3 VH3-13-21 1.0% 46 BUR 119 3 VH3-13-7 21 21.4% 67 s4D5 107 3 VH3-11-3 1 1.0% 4619 116 3 VH3-13-16 4 4.1% 118 s5D4 99 3 VH3-13-1 0 0.0% 46 s6A8 100 3VH3-13-1 0 0.0% 46 HIV-loop13 123 3 VH3-13-12 17 17.3% 12 TR1.32 112 3VH3-11-8 18 18.6% 88 L2B10 97 3 VH3-11-3 1 1.0% 46 TR1.5 114 3 VH3-11-821 21.6% 88 s6H9 101 3 VH3-13-25 0 0.0% 46 8 112 3 VH3-13-1 6 6.1% 11823 115 3 VH3-13-1 6 6.1% 118 7 115 3 VH3-13-1 4 4.1% 118 TR1.3 120 3VH3-11-8 20 20.6% 88 18/2 125 3 VH3-13-10 0 0.0% 32 18/9 125 3 VH3-13-100 0.0% 31 30P1 119 3 VH3-13-10 0 0.0% 106 HF2-1/17 125 3 VH3-13-10 00.0% 8 A77 109 3 VH3-13-10 0 0.0% 44 B19.7 108 3 VH3-13-10 0 0.0% 44 M43119 3 VH3-13-10 0 0.0% 103 1/17 125 3 VH3-13-10 0 0.0% 31 18/17 125 3VH3-13-10 0 0.0% 31 E54 3.4 109 3 VH3-13-10 0 0.0% 26 LAMBDA-VH26 98 3VH3-13-10 1 1.0% 95 E54 3.8 111 3 VH3-13-10 1 1.0% 26 GL16 106 3VH3-13-10 1 1.0% 44 4G12 125 3 VH3-13-10 1 1.0% 56 A73 106 3 VH3-13-10 22.0% 44 AL1.3 111 3 VH3-13-10 3 3.1% 117 3.A290 118 3 VH3-13-10 2 2.0%108 Ab18 127 3 VH3-13-8 2 2.0% 100 E54 3.3 105 3 VH3-13-10 3 3.1% 2635G6 121 3 VH3-13-10 3 3.1% 57 A95 107 3 VH3-13-10 5 5.1% 44 Ab25 128 3VH3-13-10 5 5.1% 100 N87 126 3 VH3-13-10 4 4.1% 77 ED8.4 99 3 VH3-13-106 6.1% 2 RF-KL1 122 3 VH3-13-10 6 6.1% 82 AL1.1 112 3 VH3-13-10 2 2.0%117 AL3.11 102 3 VH3-13-10 1 1.0% 117 32.B9 127 3 VH3-13-8 6 6.1% 129TK1 109 3 VH3-13-10 2 2.0% 117 POP 123 3 VH3-13-10 8 8.2% 115 9F2H 127 3VH3-13-10 9 9.2% 127 VD 115 3 VH3-13-10 9 9.2% 10 Vh38Cl.10 121 3VH3-13-10 8 8.2% 74 Vh38Cl.9 121 3 VH3-13-10 8 8.2% 74 Vh38Cl.8 121 3VH3-13-10 8 8.2% 74 63P1 120 3 VH3-11-8 0 0.0% 104 60P2 117 3 VH3-11-8 00.0% 104 AL3.5 90 3 VH3-13-10 2 2.0% 117 GF4/1.1 123 3 VH3-13-10 1010.2% 39 Ab21 126 3 VH3-13-10 12 12.2% 100 TD d Vp 118 3 VH3-13-17 22.0% 16 Vh38Cl.4 119 3 VH3-13-10 8 8.2% 74 Vh38Cl.5 119 3 VH3-13-10 88.2% 74 AL3.4 104 3 VH3-13-10 1 1.0% 117 FOG1-A3 115 3 VH3-13-19 2 2.0%42 HA3D1 117 3 VH3-13-21 1 1.0% 81 E54 3.2 112 3 VH3-13-24 0 0.0% 26mAb52 128 3 VH3-13-12 2 2.0% 51 mAb53 128 3 VH3-13-12 2 2.0% 51 mAb56128 3 VH3-13-12 2 2.0% 51 mAb57 128 3 VH3-13-12 2 2.0% 51 mAb58 128 3VH3-13-12 2 2.0% 51 mAb59 128 3 VH3-13-12 2 2.0% 51 mAb105 128 3VH3-13-12 2 2.0% 51 mAb107 128 3 VH3-13-12 2 2.0% 51 E55 3.14 110 3VH3-13-19 0 0.0% 26 F13-28 106 3 VH3-13-19 1 1.0% 94 mAb55 127 3VH3-13-18 4 4.1% 51 YSE 117 3 VH3-13-24 6 6.1% 72 E55 3.23 106 3VH3-13-19 2 2.0% 26 RF-TS5 101 3 VH3-13-1 3 3.1% 85 N42P5 124 3 VH3-13-27 7.1% 77 FOG1-H6 110 3 VH3-13-16 7 7.1% 42 O-81 115 3 VH3-13-19 1111.2% 47 HIV-s8 122 3 VH3-13-12 11 11.2% 12 mAb114 125 3 VH3-13-19 1212.2% 71 33.F12 116 3 VH3-13-2 4 4.1% 129 4B4 119 3 VH3-1X-3 0 0.0% 101M26 123 3 VH3-1X-3 0 0.0% 103 VHGL 3.1 100 3 VH3-1X-3 0 0.0% 26 E55 3.13113 3 VH3-1X-3 1 1.0% 26 SB5/D6 101 3 VH3-1X-6 3 3.0% 2 RAY4 101 3VH3-1X-6 3 3.0% 2 82-D V-D 106 3 VH3-1X-3 5 5.0% 112 MAL 129 3 VH3-1X-35 5.0% 72 LOC 123 3 VH3-1X-6 5 5.0% 72 LSF2 101 3 VH3-1X-6 11 11.0% 2HIB RC3 100 3 VH3-1X-6 11 11.0% 1 56P1 119 3 VH3-13-7 0 0.0% 104 M72 1223 VH3-13-7 0 0.0% 103 M74 121 3 VH3-13-7 0 0.0% 103 E54 3.5 105 3VH3-13-7 0 0.0% 26 2E7 123 3 VH3-13-7 0 0.0% 63 2P1 117 3 VH3-13-7 00.0% 104 RF-SJ2 127 3 VH3-13-7 1 1.0% 83 PR-TS1 114 3 VH3-13-7 1 1.0% 85KIM46H 127 3 VH3-13-13 0 0.0% 18 E55 3.6 108 3 VH3-13-7 2 2.0% 26 E553.10 107 3 VH3-13-13 1 1.0% 26 3.B6 114 3 VH3-13-13 1 1.0% 108 E54 3.6110 3 VH3-13-13 1 1.0% 26 FL2-2 114 3 VH3-13-13 1 1.0% 80 RF-SJ3 112 3VH3-13-7 2 2.0% 85 E55 3.5 105 3 VH3-13-14 1 1.0% 26 BSA3 121 3VH3-13-13 1 1.0% 73 HMST-1 119 3 VH3-13-7 3 3.1% 130 RF-TS2 126 3VH3-13-13 4 4.1% 82 E55 3.12 109 3 VH3-13-15 0 0.0% 26 19.E7 126 3VH3-13-14 3 3.1% 129 11-50 119 3 VH3-13-13 6 6.1% 130 E29.1 120 3VH3-13-15 2 2.0% 25 E55 3.16 108 3 VH3-13-7 6 6.1% 26 TNF-E1 117 3VH3-13-7 7 7.1% 42 RF-SJ1 127 3 VH3-13-13 6 6.1% 83 FOG1-A4 116 3VH3-13-7 8 8.2% 42 TNF-A1 117 3 VH3-13-15 4 4.1% 42 PR-SJ2 107 3VH3-13-14 8 8.2% 85 HN.14 124 3 VH3-13-13 10 10.2% 33 CAM′ 121 3VH3-13-7 12 12.2% 65 HIV-B8 125 3 VH3-13-7 9 9.2% 12 HIV-b27 125 3VH3-13-7 9 9.2% 12 HIV-b8 125 3 VH3-13-7 9 9.2% 12 HIV-s4 125 3 VH3-13-79 9.2% 12 HIV-B26 125 3 VH3-13-7 9 9.2% 12 HIV-B35 125 3 VH3-13-7 1010.2% 12 HIV-b18 125 3 VH3-13-7 10 10.2% 12 HIV-b22 125 3 VH3-13-7 1111.2% 12 HIV-b13 125 3 VH3-13-7 12 12.2% 12 333 117 3 VH3-14-4 24 24.0%24 1H1 120 3 VH3-14-4 24 24.0% 24 1B11 120 3 VH3-14-4 23 23.0% 24 CLL3O2-3 86 3 VH3-13-19 1 1.0% 29 GA 110 3 VH3-13-7 19 19.4% 36 JeB 99 3VH3-13-14 3 3.1% 7 GAL 110 3 VH3-13-19 10 10.2% 126 K6H6 119 3 VH3-1X-618 18.0% 60 K4B8 119 3 VH3-1X-6 18 18.0% 60 K5B8 119 3 VH3-1X-6 18 18.0%60 K5C7 119 3 VH3-1X-6 19 19.0% 60 K5G5 119 3 VH3-1X-6 19 19.0% 60 K6F5119 3 VH3-1X-6 19 19.0% 60 AL3.16 98 3 VH3-13-10 1 1.0% 117 N86P2 98 3VH3-13-10 3 3.1% 77 N54P6 95 3 VH3-13-16 7 7.1% 77 LAMBDA HT112-1 126 4VH4-11-2 0 0.0% 3 HY18 121 4 VH4-11-2 0 0.0% 43 mAb63 126 4 VH4-11-2 00.0% 45 FS-3 105 4 VH4-11-2 0 0.0% 86 FS-5 111 4 VH4-11-2 0 0.0% 86 FS-7107 4 VH4-11-2 0 0.0% 86 FS-8 110 4 VH4-11-2 0 0.0% 86 PR-TS2 105 4VH4-11-2 0 0.0% 85 RF-TMC 102 4 VH4-11-2 0 0.0% 85 mAb216 122 4 VH4-11-21 1.0% 15 mAb410.7.F91 122 4 VH4-11-2 1 1.0% 52 mAbA6H4C5 124 4 VH4-11-21 1.0% 15 Ab44 127 4 VH4-11-2 2 2.1% 100 6H-3C4 124 4 VH4-11-2 3 3.1% 59FS-6 108 4 VH4-11-2 6 6.2% 86 FS-2 114 4 VH4-11-2 6 6.2% 84 HIG1 126 4VH4-11.2 7 7.2% 62 FS-4 105 4 VH4-11-2 8 8.2% 86 SA-4A 123 4 VH4-11-2 99.3% 125 LES-C 119 4 VH4-11-2 10 10.3% 99 DI 78 4 VH4-11-9 16 16.5% 58Ab26 126 4 VH4-31-4 8 8.1% 100 TS2 124 4 VH4-31-12 15 15.2% 110 265-695115 4 VH4-11-7 16 16.5% 5 WAH 129 4 VH4-31-13 19 19.2% 93 268-D 122 4VH4-11-8 22 22.7% 6 58P2 118 4 VH4-11-8 0 0.0% 104 mAb67 128 4 VH4-21-41 1.0% 45 4.L39 115 4 VH4-11-8 2 2.1% 108 mF7 111 4 VH4-31-13 3 3.0% 7533.C9 122 4 VH4-21-5 7 7.1% 129 Pag-1 124 4 VH4-11-16 5 5.2% 50 B3 123 4VH4-21-3 8 8.2% 53 IC4 120 4 VH4-11-8 6 6.2% 70 C6B2 127 4 VH4-31-12 44.0% 48 N78 118 4 VH4-11-9 11 11.3% 77 B2 109 4 VH4-11-8 12 12.4% 53WRD2 123 4 VH4-11-12 6 6.2% 90 mAb426.4.2F20 126 4 VH4-11-8 2 2.1% 52E54 4.58 115 4 VH4-11-8 1 1.0% 26 WRD6 123 4 VH4-11-12 10 10.3% 90mAb426.12.3F1.4 122 4 VH4-11-9 4 4.1% 52 E54 4.2 108 4 VH4-21-6 2 2.0%26 WIL 127 4 VH4-31-13 0 0.0% 90 COF 126 4 VH4-31-13 0 0.0% 90 LAR 122 4VH4-31-13 2 2.0% 90 WAT 125 4 VH4-31-13 4 4.0% 90 mAb61 123 4 VH4-31-135 5.1% 45 WAG 127 4 VH4-31-4 0 0.0% 90 RF-SJ4 108 4 VH4-31-12 2 2.0% 85E54 4.4 110 4 VH4-11-7 0 0.0% 26 E55 4.A1 108 4 VH4-11-7 0 0.0% 26PR-SJ1 103 4 VH4-11-7 1 1.0% 85 E54 4.23 111 4 VH4-11-7 1 1.0% 26 CLL77-2 97 4 VH4-11-12 0 0.0% 29 37P1 95 4 VH4-11-12 0 0.0% 104 ALL52 30-291 4 VH4-31-12 4 4.0% 29 EBV-21 98 5 VH5-12-1 0 0.0% 13 CB-4 98 5VH5-12-1 0 0.0% 13 CLL-12 98 5 VH5-12-1 0 0.0% 13 L3-4 98 5 VH5-12-1 00.0% 13 CLL11 98 5 VH5-12-1 0 0.0% 17 CORD3 98 5 VH5-12-1 0 0.0% 17CORD4 98 5 VH5-12-1 0 0.0% 17 CORD8 98 5 VH5-12-1 0 0.0% 17 CORD9 98 5VH5-12-1 0 0.0% 17 CD+1 98 5 VH5-12-1 0 0.0% 17 CD+3 98 5 VH5-12-1 00.0% 17 CD+4 98 5 VH5-12-1 0 0.0% 17 CD−1 98 5 VH5-12-1 0 0.0% 17 CD−598 5 VH5-12-1 0 0.0% 17 VERG14 98 5 VH5-12-1 0 0.0% 17 PBL1 98 5VH5-12-1 0 0.0% 17 PBL10 98 5 VH5-12-1 0 0.0% 17 STRAb SA-1A 127 5VH5-12-1 0 0.0% 125 DOB′ 122 5 VH5-12-1 0 0.0% 97 VERG5 98 5 VH5-12-1 00.0% 17 PBL2 98 5 VH5-12-1 1 1.0% 17 Tu16 119 5 VH5-12-1 1 1.0% 49 PBL1298 5 VH5-12-1 1 1.0% 17 CD+2 98 5 VH5-12-1 1 1.0% 17 CORD10 98 5VH5-12-1 1 1.0% 17 PBL9 98 5 VH5-12-1 1 1.0% 17 CORD2 98 5 VH5-12-1 22.0% 17 PBL6 98 5 VH5-12-1 2 2.0% 17 CORD5 98 5 VH5-12-1 2 2.0% 17 CD−298 5 VH5-12-1 2 2.0% 17 CORD1 98 5 VH5-12-1 2 2.0% 17 CD−3 98 5 VH5-12-13 3.1% 17 VERG4 98 5 VH5-12-1 3 3.1% 17 PBL13 98 5 VH5-12-1 3 3.1% 17PBL7 98 5 VH5-12-1 3 3.1% 17 HAN 119 5 VH5-12-1 3 3.1% 97 VERG3 98 5VH5-12-1 3 3.1% 17 PBL3 98 5 VH5-12-1 3 3.1% 17 VERG7 98 5 VH5-12-1 33.1% 17 PBL5 94 5 VH5-12-1 0 0.0% 17 CD−4 98 5 VH5-12-1 4 4.1% 17 CLL1098 5 VH5-12-1 4 4.1% 17 PBL11 98 5 VH5-12-1 4 4.1% 17 CORD6 98 5VH5-12-1 4 4.1% 17 VERG2 98 5 VH5-12-1 5 5.1% 17 83P2 119 5 VH5-12-1 00.0% 103 VERG9 98 5 VH5-12-1 6 6.1% 17 CLL6 98 5 VH5-12-1 6 6.1% 17 PBL898 5 VH5-12-1 7 7.1% 17 Ab2022 120 5 VH5-12-1 3 3.1% 100 CAV 127 5VH5-12-4 0 0.0% 97 HOW′ 120 5 VH5-12-4 0 0.0% 97 PET 127 5 VH5-12-4 00.0% 97 ANG 121 5 VH5-12-4 0 0.0% 97 KER 121 5 VH5-12-4 0 0.0% 97 5.M13118 5 VH5-12-4 0 0.0% 107 Au2.1 118 5 VH5-12-4 1 1.0% 49 WS1 126 5VH5-12-1 9 9.2% 110 TD Vn 98 5 VH5-12-4 1 1.0% 16 TEL13 116 5 VH5-12-1 99.2% 73 E55 5.237 112 5 VH5-12-4 2 2.0% 26 VERG1 98 5 VH5-12-1 10 10.2%17 CD4-74 117 5 VH5-12-1 10 10.2% 42 257-D 125 5 VH5-12-1 11 11.2% 6CLL4 98 5 VH5-12-1 11 11.2% 17 CLL8 98 5 VH5-12-1 11 11.2% 17 Ab2 124 5VH5-12-1 12 12.2% 120 Vh383ex 98 5 VH5-12-1 12 12.2% 120 CLL3 98 5VH5-12-2 11 11.2% 17 Au59.1 122 5 VH5-12-1 12 12.2% 49 TEL16 117 5VH5-12-1 12 12.2% 73 M61 104 5 VH5-12-1 0 0.0% 103 Tu0 99 5 VH5-12-1 55.1% 49 P2-51 122 5 VH5-12-1 13 13.3% 121 P2-54 122 5 VH5-12-1 11 11.2%121 P1-56 119 5 VH5-12-1 9 9.2% 121 P2-53 122 5 VH5-12-1 10 10.2% 121P1-51 123 5 VH5-12-1 19 19.4% 121 P1-54 123 5 VH5-12-1 3 3.1% 121 P3-69127 5 VH5-12-1 4 4.1% 121 P3-9 119 5 VH5-12-1 4 4.1% 121 1-185-37 125 5VH5-12-4 0 0.0% 124 1-187-29 125 5 VH5-12-4 0 0.0% 124 P1-58 128 5VH5-12-4 10 10.2% 121 P2-57 118 5 VH5-12-4 3 3.1% 121 P2-55 123 5VH5-12-1 5 5.1% 121 P2-56 123 5 VH5-12-1 20 20.4% 121 P2-52 122 5VH5-12-1 11 11.2% 121 P3-60 122 5 VH5-12-1 8 8.2% 121 P1-57 123 5VH5-12-1 4 4.1% 121 P1-55 122 5 VH5-12-1 14 14.3% 121 MD3-4 128 5VH5-12-4 12 12.2% 5 P1-52 121 5 VH5-12-1 11 11.2% 121 CLL5 98 5 VH5-12-113 13.3% 17 CLL7 98 5 VH5-12-1 14 14.3% 17 L2F10 100 5 VH5-12-1 1 1.0%46 L3B6 98 5 VH5-12-1 1 1.0% 46 VH6.A12 119 6 VH6-35-1 13 12.9% 122 s5A9102 6 VH6-35-1 1 1.0% 46 s6G4 99 6 VH6-35-1 1 1.0% 46 ss3 99 6 VH6-35-11 1.0% 46 6-1G1 101 6 VH6-35-1 0 0.0% 14 F19L16 107 6 VH6-35-1 0 0.0% 68L16 120 6 VH6-35-1 0 0.0% 69 M71 121 6 VH6-35-1 0 0.0% 103 ML1 120 6VH6-35-1 0 0.0% 69 F19ML1 107 6 VH6-35-1 0 0.0% 68 15P1 127 6 VH6-35-1 00.00% 104 VH6.N1 121 6 VH6-35-1 0 0.0% 122 VH6.N11 123 6 VH6-35-1 0 0.0%122 VH6.N12 123 6 VH6-35-1 0 0.0% 122 VH6.N2 125 6 VH6-35-1 0 0.0% 122VH6.N5 125 6 VH6-35-1 0 0.0% 122 VH6.N6 127 6 VH6-35-1 0 0.0% 122 VH6.N7126 6 VH6-35-1 0 0.0% 122 VH6.N8 123 6 VH6-35-1 0 0.0% 122 VH6.N9 123 6VH6-35-1 0 0.0% 122 VH6.N10 123 6 VH6-35-1 0 0.0% 122 VH6.A3 123 6VH6-35-1 0 0.0% 122 VH6.A1 124 6 VH6-35-1 0 0.0% 122 VH6.A4 120 6VH6-35-1 0 0.0% 122 E55 6.16 116 6 VH6-35-1 0 0.0% 26 E55 6.17 120 6VH6-35-1 0 0.0% 26 E55 6.6 120 6 VH6-35-1 0 0.0% 26 VHGL 6.3 102 6VH6-35-1 0 0.0% 26 CB-201 118 6 VH6-35-1 0 0.0% 109 VH6.N4 122 6VH6-35-1 0 0.0% 122 E54 6.4 109 6 VH6-35-1 1 1.0% 26 VH6.A6 126 6VH6-35-1 1 1.0% 122 E55 6.14 120 6 VH6-35-1 1 1.0% 26 E54 6.6 107 6VH6-35-1 1 1.0% 26 E55 6.10 112 6 VH6-35-1 1 1.0% 26 E54 6.1 107 6VH6-35-1 2 2.0% 26 E55 6.13 120 6 VH6-35-1 2 2.0% 26 E55 6.3 120 6VH6-35-1 2 2.0% 26 E55 6.7 116 6 VH6-35-1 2 2.0% 26 E55 6.2 120 6VH6-35-1 2 2.0% 26 E55 6.X 111 6 VH6-35-1 2 2.0% 26 E55 6.11 111 6VH6-35-1 3 3.0% 26 VH6.A11 118 6 VH6-35-1 3 3.0% 122 A10 107 6 VH6-35-13 3.0% 68 E55 6.1 120 6 VH6-35-1 4 4.0% 26 FK-001 124 6 VH6-35-1 4 4.0%65 VH6.A5 121 6 VH6-35-1 4 4.0% 122 VH6.A7 123 6 VH6-35-1 4 4.0% 122HBp2 119 6 VH6-35-1 4 4.0% 4 Au46.2 123 6 VH6-35-1 5 5.0% 49 A431 106 6VH6-35-1 5 5.0% 68 VH6.A2 120 6 VH6-35-1 5 5.0% 122 VH6.A9 125 6VH6-35-1 8 7.9% 122 VH6.A8 118 6 VH6-35-1 10 9.9% 122 VH6-FF3 118 6VH6-35-1 2 2.0% 123 VH6.A10 126 6 VH6-35-1 12 11.9% 122 VH6-EB10 117 6VH6-35-1 3 3.0% 123 VH6-E6 119 6 VH6-35-1 6 5.9% 123 VH6-FE2 121 6VH6-35-1 6 5.9% 123 VH6-EE6 116 6 VH6-35-1 6 5.9% 123 VH6-FD10 118 6VH6-35-1 6 5.9% 123 VH6-EX8 113 6 VH6-35-1 6 5.9% 123 VH6-FG9 121 6VH6-35-1 8 7.9% 123 VH6-E5 116 6 VH6-35-1 9 8.9% 123 VH6-EC8 122 6VH6-35-1 9 8.9% 123 VH6-E10 120 6 VH6-35-1 10 9.9% 123 VH6-FF11 122 6VH6-35-1 11 10.9% 123 VH6-FD2 115 6 VH6-35-1 11 10.9% 123 CLL10 17-2 886 VH6-35-1 4 4.0% 29 VH6-BB11 94 6 VH6-35-1 4 4.0% 123 VH6-B4I 93 6VH6-35-1 7 6.9% 123 JU17 102 6 VH6-35-1 3 3.0% 114 VH6-BD9 96 6 VH6-35-111 10.9% 123 VH6-BB9 94 6 VH6-35-1 12 11.9% 123

TABLE 3A assignment of rearranged V kappa sequences to their germlinecounterparts Family¹ Name Rearranged² Sum 1 Vk1-1 28 1 Vk1-2 0 1 Vk1-3 11 Vk1-4 0 1 Vk1-5 7 1 Vk1-6 0 1 Vk1-7 0 1 Vk1-8 2 1 Vk1-9 9 1 Vk1-10 0 1Vk1-11 1 1 Vk1-12 7 1 Vk1-13 1 1 Vk1-14 7 1 Vk1-15 2 1 Vk1-16 2 1 Vk1-1716 1 Vk1-18 1 1 Vk1-19 33 1 Vk1-20 1 1 Vk1-21 1 1 Vk1-22 0 1 Vk1-23 0119 entries 2 Vk2-1 0 2 Vk2-2 1 2 Vk2-3 0 2 Vk2-4 0 2 Vk2-5 0 2 Vk2-6 162 Vk2-7 0 2 Vk2-8 0 2 Vk2-9 1 2 Vk2-10 0 2 Vk2-11 7 2 Vk2-12 0  25entries 3 Vk3-1 1 3 Vk3-2 0 3 Vk3-3 35 3 Vk3-4 115 3 Vk3-5 0 3 Vk3-6 0 3Vk3-7 1 3 Vk3-8 40 192 entries 4 Vk4-1 33  33 entries 5 Vk5-1 1  1 entry6 Vk6-1 0 6 Vk6-2 0  0 entries 7 Vk7-1 0  0 entries

TABLE 3B assignment of rearranged V lambda sequences to their germlinecounterparts Family¹ Name Rearranged² Sum 1 DPL1 1 1 DPL2 14 1 DPL3 6 1DPL4 1 1 HUMLV117 4 1 DPL5 13 1 DPL6 0 1 DPL7 0 1 DPL8 3 1 DPL9 0 42entries 2 DPL10 5 2 VLAMBDA 2.1 0 2 DPL11 23 2 DPL12 15 2 DPL13 0 2DPL14 0 43 entries 3 DPL16 10 3 DPL23 19 3 Humlv318 9 38 entries 7 DPL181 7 DPL19 0  1 entries 8 DPL21 2 8 HUMLV801 6  8 entries 9 DPL22 0  0entries unassigned DPL24 0  0 entries 10  gVLX-4.4 0  0 entries

TABLE 3C assignment of rearranged V heavy chain sequences to theirgermline counterparts Family¹ Name Rearranged² Sum 1 VH1-12-1 38 1VH1-12-8 2 1 VH1-12-2 2 1 VH1-12-9 2 1 VH1-12-3 0 1 VH1-12-4 0 1VH1-12-5 3 1 VH1-12-6 0 1 VH1-12-7 23 1 VH1-13-1 1 1 VH1-13-2 1 1VH1-13-3 0 1 VH1-13-4 0 1 VH1-13-5 0 1 VH1-13-6 17 1 VH1-13-7 0 1VH1-13-8 3 1 VH1-13-9 0 1 VH1-13-10 0 1 VH1-13-11 0 1 VH1-13-12 10 1VH1-13-13 0 1 VH1-13-14 0 1 VH1-13-15 4 1 VH1-13-16 2 1 VH1-13-17 0 1VH1-13-18 1 1 VH1-13-19 0 1 VH1-1X-1 1 110 entries 2 VH2-21-1 0 2VH2-31-1 0 2 VH2-31-2 1 2 VH2-31-3 1 2 VH2-31-4 0 2 VH2-31-5 2 2VH2-31-6 0 2 VH2-31-7 0 2 VH2-31-14 1 2 VH2-31-8 0 2 VH2-31-9 0 2VH2-31-10 0 2 VH2-31-11 1 2 VH2-31-12 0 2 VH2-31-13 1  7 entries 3VH3-11-1 0 3 VH3-11-2 0 3 VH3-11-3 5 3 VH3-11-4 0 3 VH3-11-5 1 3VH3-11-6 1 3 VH3-11-7 0 3 VH3-11-8 5 3 VH3-13-1 9 3 VH3-13-2 3 3VH3-13-3 0 3 VH3-13-4 0 3 VH3-13-5 0 3 VH3-13-6 0 3 VH3-13-7 32 3VH3-13-8 4 3 VH3-13-9 0 3 VH3-13-10 46 3 VH3-13-11 0 3 VH3-13-12 11 3VH3-13-13 17 3 VH3-13-14 8 3 VH3-13-15 4 3 VH3-13-16 3 3 VH3-13-17 2 3VH3-13-18 1 3 VH3-13-19 13 3 VH3-13-20 1 3 VH3-13-21 1 3 VH3-13-22 0 3VH3-13-23 0 3 VH3-13-24 4 3 VH3-13-25 1 3 VH3-13-26 6 3 VH3-14-1 1 3VH3-14-4 15 3 VH3-14-2 0 3 VH3-14-3 0 3 VH3-1X-1 0 3 VH3-1X-2 0 3VH3-1X-3 6 3 VH3-1X-4 0 3 VH3-1X-5 0 3 VH3-1X-6 11 3 VH3-1X-7 0 3VH3-1X-8 1 3 VH3-1X-9 0 212 entries 4 VH4-11-1 0 4 VH4-11-2 20 4VH4-11-3 0 4 VH4-11-4 0 4 VH4-11-5 0 4 VH4-11-6 0 4 VH4-11-7 5 4VH4-11-8 7 4 VH4-11-9 3 4 VH4-11-10 0 4 VH4-11-11 0 4 VH4-11-12 4 4VH4-11-13 0 4 VH4-11-14 0 4 VH4-11-15 0 4 VH4-11-16 1 4 VH4-21-1 0 4VH4-21-2 0 4 VH4-21-3 1 4 VH4-21-4 1 4 VH4-21-5 1 4 VH4-21-6 1 4VH4-21-7 0 4 VH4-21-8 0 4 VH4-21-9 0 4 VH4-31-1 0 4 VH4-31-2 0 4VH4-31-3 0 4 VH4-31-4 2 4 VH4-31-5 0 4 VH4-31-6 0 4 VH4-31-7 0 4VH4-31-8 0 4 VH4-31-9 0 4 VH4-31-10 0 4 VH4-31-11 0 4 VH4-31-12 4 4VH4-31-13 7 4 VH4-31-14 0 4 VH4-31-15 0 4 VH4-31-16 0 4 VH4-31-17 0 4VH4-31-18 0 4 VH4-31-19 0 4 VH4-31-20 0  57 entries 5 VH5-12-1 82 5VH5-12-2 1 5 VH5-12-3 0 5 VH5-12-4 14  97 entries 6 VH6-35-1 74  74entries

TABLE 4A Analysis of V kappa subgroup 1 Framework I amino acid¹ 1 2 3 45 6 7 8 9 10 11 12 13 14 15 16 A  1  1 102   1 B  1  1 C  1 D 64 E  8 14 1 F  1  6  1 G 105  H I 65  4 K  1 L  6 21 96  1 M  1 66 N P 103   1  2 1 Q 62 88  1 R S 89 102  80 103  103  T  1 88 18 V  1  9  8  2 98 W X 1 Y — unknown (?) not sequenced 31 31 18 18 17 16 16  2  1 sum of seq²74 74 87 87 88 89 89 103  104  105  105  105  105  105  105  105 oomcaa³ 64 65 62 66 88 88 89 103  102  80 96 103  102  103  98 105 mcaa⁴ D I Q M T Q S P S S L S A S V G rel. oomcaa⁵ 86% 88% 71% 76% 100%99% 100% 100% 98% 76% 91% 98% 97% 98% 93% 100% pos occupied⁶  4  5  5  2 1  2  1  1  3  4  3  2  3  3  5  1 Framework I CDR I amino acid¹ 17 1819 20 21 22 23 24 25 26 27 A B C D A  1  1  1 103  B  1 C 105  D 101  E 2  1  1  2 F  2 G  1 H  1 I  6  4 101   1 K  2  1 L  1 M N  1 P Q 20100  R 94 81 S  5  1 102  T  6 99 103   1  1 V 98  2 W X  1 Y  1 — 105 105  105  105  unknown (?) not sentenced sum of seq² 105  105  105  105 105  105  105  105  105  105  105  105  105  105  105  oomcaa³ 101  9498 99 101  103  105  81 103  102  100  105  105  105  105  mcaa⁴ D R V TI T C R A S Q — — — — rel. oomcaa⁵ 96% 90% 93% 94% 96% 98% 100% 77% 98%97% 95% 100% 100% 100% 100% pos occupied⁶  4  3  3  4  3  3  1  5  3  4 5  1  1  1  1 CDR I Framework II amino acid¹ E F 28 29 30 31 32 33 3435 36 37 38 39 40 A  1  1  1 42 B  1  1 C  1 D 25  1  5  7  1 E  1  2 F 1  1  7  6 G 25  7  3  4 H  1  2  2  1  2 I 98  1  4  1 K  7 95 L  2  1101  M N  6 16 42 50 P 102  Q 98 103   2 R 16  3  2  3  1 S 41  2 57 32 3  1  1  1 T  7  4  4  1 V  1  4  1  1 W 21 104  X  1 Y  1 60 98 — 105 105  unknown (?)  3 not sequenced  1  1  1  1  1  1  1  1  1  1 sum ofseq² 105  105  105  105  105  104  104  104  104  104  104  104  104 104  104  oomcaa³ 105  105  41 98 57 42 60 101  50 104  98 98 103  95102  mcaa⁴ — — S I S N Y L N W Y Q Q K P rel. oomcaa⁵ 100% 100% 39% 93%54% 40% 58% 97% 48% 100% 94% 94% 99% 91% 98% pos occupied⁶  1  1  6  412 11  9  4  8  1  2  5  2  4  3 Framework II CDR II amino acid¹ 41 4243 44 45 46 47 48 49 50 51 52 53 54 55 A 94 50 95 B C D 21  1  1  1 E  1 3  1  1  1  1 33 F  1  3  1 G 100   1  9  2 H  2  1 I  1  1 100   1 K95 86 16  2  5 L  1 89 103  101  M  2 N 10  2  1 25 P 104   1  1 Q  1  162 R  3  3  1  1  2 S  1  5  1  1 99 41  2 T  3  1  1  4  1 31 V  9  9 1  1 W X  1  1 Y 92  1 — unknown (?)  3 not sequenced  1  1  1  1  1  1 2  3  3  2  1  1  1  1  1 sum of seq² 104  104  104  104  104  104 103  102  102  103  104  104  104  104  104  oomcaa³ 100  95 94 104  8689 103  100  92 50 95 99 41 101  62 mcaa⁴ G K A P K L L I Y A A S S L Qrel. oomcaa⁵ 96% 91% 90% 100% 83% 86% 100% 98% 90% 49% 91% 95% 39% 97%60% pos occupied⁶  2  6  3  1  8  6  1  2  4 10  6  6  9  3  6 CDR IIFramework III amino acid¹ 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 A 3  2  1  1  1 B  1 C D  1 67 E  1 30 F  1 103   3 G  2 105  105   4101  102  H  3 I  3  4  1  3 K  1  1  1 L  1 M  1 N  6 P  1 101   2 Q  1R  1 103   1  1  1  2 S 68  2 103  98 96 100  T 19  1  1  2  3 101  V 99 1  1 W X  1  1  1  2 Y  1  1 — unknown (?) not sequenced sum of seq²105  105  105  105  105  105  105  105  105  105  105  105  105  105 105  oomcaa³ 68 105  99 101  103  103  103  98 105  96 101  100  102 101  67 mcaa⁴ S G V P S R F S G S G S G T D rel. oomcaa⁵ 65% 100% 94%96% 98% 98% 98% 93% 100% 91% 96% 95% 97% 96% 64% pos occupied⁶ 10  1  4 4  2  3  3  5  1  5  4  4  4  4  7 Framework III amino acid¹ 71 72 7374 75 76 77 78 79 80 81 82 83 84 85 A  3  1  2 101   1 B  1  3  2 C D  116 101  E 83 F 102   1 21 73 G  4  1  2 H I 99  5 17 K L 81 103   1  1 M 1 N  7  4  1 P 97  1 Q 97 R  2  1  2 S  2  1 86 94  4  1 T 98 102   2 1 97 V  1  2  4  1 11  1 W X  1  1  2 Y  1 — unknown (?) not sequenced 1  1  1  1  1  1  1  1  2  2  2  2  2  2  3 sum ofseq² 104  104  104 104  104  104  104  104  103  103  103  103  103  103  102  oomcaa³ 102 98 81 102  99 86 94 103  97 97 83 101  73 101  97 mcaa⁴ F T L T I S S LQ P E D F A T rel. oomcaa⁵ 98% 94% 78% 98% 95% 83% 90% 99% 94% 94% 81%98% 71% 98% 95% pos occupied⁶  3  4  3  3  3  7  5  2  4  3  5  2  5  2 6 Framework III CDR III amino acid¹ 86 87 88 89 90 91 92 93 94 95 A B CD E F A  1  7  1  5  1 B  2  3 C 102  D 23  5  1 E  1  1  1  1 F  7  313 G  1  1  2  1  1 H  1  4  6  7  3  1 I  4  1  2  1 K  1  7  1 L  7  6 2 18  2 M N  6 31 19  1 P  1 82  6 Q 90 86  1  2 R  1  2  2 S  1 27  358  5 10 T  3  1 15 25 V  5 W  1 X Y 101  93 42 32  1 23 —  3 82 88 8989 89 89 unknown (?)  1 not sequenced  2  3  3  2  2  1  1  1  1  4 1616 16 16 16 16 sum of seq² 103  102  102  103  103  104  104  104  104 101  89 89 89 89 89 89 oomcaa³ 101  93 102  90 86 42 32 58 25 82 82 8889 89 89 89 mcaa⁴ Y Y C Q Q Y Y S T P — — — — — — rel. oomcaa⁵ 98% 91%100% 87% 83% 40% 31% 56% 24% 81% 92% 99% 100% 100% 100% 100% posoccupied⁶  3  3  1  4  5 11 12 10 14  8  3  2  1  1  1  1 CDR IIIFramework IV amino acid¹ 96 97 98 99 100 101 102 103 104 105 106 A 107108 sum A  1 627 B  1  1 19 C 209 D  1 15 459 E  2 65 258 F  6 86  2 451G 87 29 87  2 894 H  2  1 40 I  5  1 72 606 K  1  1 77 79 480 L 18  1  122  4  2 793 M  1  5 77 N  1  1  2 232 P  6  7  1 620 Q  1 48  1 865 R 6  6  2 70 413 S  2  2 1636 T  2 82 87  3  2 1021 V  2  1 63  3 440 W15 141 X 14 Y 16 564 —  4  1 85  1 1250 unknown (?) 7 not sequenced 1616 18 18 18 18 18 18 19 19 20 20 20 31 589 sum of seq² 89 89 87 87 87 8787 87 86 86 85 85 85 74 oomcaa³ 18 82 86 87 48 87 87 77 63 65 72 85 7970 mcaa⁴ L T F G G G T K V E I — K R rel. oomcaa⁵ 20% 92% 99% 100% 55%100% 100% 89% 73% 76% 85% 100% 93% 95% pos occupied⁶ 17  7  2  1  5  1 1  4  3  5  6  1  4  4

TABLE 4B Analysis of V kappa subgroup 2 Framework I amino acid¹ 1 2 3 45 6 7 8 9 10 11 12 13 14 15 16 A B C D 14 E  3 F  1  1 G 22 H I  8 K L 3  1 17 18  6 M 15 N P 18 18 15 Q 18 R S 18 17 T 17 21 V  6 17  1 18 XY — unknown (?)  1 not sequenced  5  5  5  5  4  4  4  4  4  4  4  4  4 1  1 sum of seq² 17 17 17 17 18 18 18 18 18 18 18 18 18 21 21 22oomcaa³ 14  8 17 15 17 18 18 18 17 17 18 18 18 21 15 22 mcaa⁴ D I V M TQ S P L S L P V T P G rel. oomcaa⁵ 82% 47% 100% 88% 94% 100% 100% 100%94% 94% 100% 100% 100% 100% 71% 100% pos occupied⁶  2  3  1  3  1  1  1 1  2  2  1  1  1  1  2  1 Framework I CDR I amino acid¹ 17 18 19 20 2122 23 24 25 26 27 A B C D E A 22 B C 22 D  1 E 15 F G  1 H 16 I 22 K  1L  1 22 13 M  1 N P 22 Q  7  1 21 R 21  2 S 22 21 22 22 22 19 T V  8 X 1 Y —  4 unknown (?) not sequenced sum of seq² 22 22 22 22 22 22 22 2222 22 22 22 22 22 22 22 oomcaa³ 15 22 22 22 22 21 22 21 22 22 21 22 2213 16 19 mcaa⁴ E P A S I S C R S S Q S L L H S rel. oomcaa⁵ 68% 100%100% 100% 100 95% 100% 95% 100% 100% 95% 100% 100% 59% 73% 86% posoccupied⁶  2  1  1  1  1  2  1  2  1  1  2  1  1  3  4  3 CDR IFramework II amino acid¹ F 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42A B C D  9  1  1 11 E F  2  7 G 22 22 H  1  1 I K  1 15 L 22 16 M N 10 7 12  9 P 22 Q  6 22 22 R  7 S  1 T  8 V W 22 X  1  1  1 Y  1 11 21 15— 22 unknown (?) not sequenced sum of seq² 22 22 22 22 22 22 22 22 22 2222 22 22 22 22 22 oomcaa³ 22 10 22 11 12 21 22 11 22 15 16 22 15 22 2222 mcaa⁴ — N G Y N Y L D W Y L Q K P G Q rel. oomcaa⁵ 100% 45% 100% 50%55% 95% 100% 50% 100% 68% 73% 100% 68% 100% 100% 100% pos occupied⁶  1 5  1  5  4  2  1  4  1  2  2  1  2  1  1  1 Framework Framework II CDRII III amino acid¹ 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 A 14B C D  7 E  1 F G 12  1 22 H I  1 22 K  5 L 14 21 14  1 M N 18 P 21 Q 12 1 R  8  7  1 22 S 21  2 22  2 22 T  1 V  1  6 22 W X Y 21  1 — unknown(?) not sequenced 1  1  1  1  1  1 sum of seq² 21 21 21 22 22 22 21 2121 22 22 22 22 22 22 22 oomcaa³ 21 21 12 14 21 22 21 14 12 22 18 22 1422 22 22 mcaa⁴ S P Q L L I Y L G S N R A S G V rel. oomcaa⁵ 100% 100%57% 64% 95% 100% 100% 67% 57% 100% 82% 100% 64% 100% 100% 100% posoccupied⁶  1  1  3  3  2  1  1  4  4  1  4  1  3  1  1  1 Framework IIIamino acid¹ 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 A B C D 22 1  1 22 E F 21 22 G 21 22 21 H I  1 K 19 L 21  1 M N P 22 Q R 20  1 S 1 22 21 22 T  1 22 21 V  1 W X Y — unknown (?)  1 not sequenced  1  1sum of seq² 22 22 22 22 22 22 22 22 22 22 22 22 22 22 21 21 oomcaa³ 2222 20 21 22 21 21 22 22 21 22 22 22 21 21 19 mcaa⁴ P D R F S G S G S G TD F T L K rel. oomcaa⁵ 100% 100% 91% 95% 100% 95% 95% 100% 100% 95% 100%100% 100% 95% 100% 90% pos occupied⁶  1  1  3  2  1  2  2  1  1  2  1  1 1  1  1  3 Framework III CDR III amino acid¹ 75 76 77 78 79 80 81 82 8384 85 86 87 88 89 90 A 20 B  1 C 21 D  1 21 E 19 20 F G  1 21 H I 21  1K L  1 M 21 N P  1 Q  1 20 R 20 S 20  1 T  1 V 21 21 19 W X Y 21 21 —unknown (?) not sequenced  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1 1 sum of seq² 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 oomcaa³21 20 20 21 19 20 20 21 21 21 19 21 21 21 21 20 mcaa⁴ I S R V E A E D VG V Y Y C M Q rel. oomcaa⁵ 100% 95% 95% 100% 90% 95% 95% 100% 100% 100%90% 100% 100% 100% 100% 95% pos occupied⁶  1  2  2  1  3  2  2  1  1  1 3  1  1  1  1  2 CDR III Framework IV amino acid¹ 91 92 93 94 95 A B CD E F 96 97 98 99 100 A 14  1 B  1 C D E F  1 17 G  6  1  2 17  2 H  1 7 I  1  3 K L 12  2  2 M N P  2 16  1  1 Q 13  1 14 R  1 S  3  2 T  8 7 17 V W  6  2 X Y  7 — 14 17 17 17 17 17 unknown (?) not sequenced 1 1  1  1  2  5  5  5  5  5  5  5  5  5  5  6 sum of seq² 21 21 21 21 2017 17 17 17 17 17 17 17 17 17 16 oomcaa³ 14 12 13  7 16 14 17 17 17 1717  7 17 17 17 14 mcaa⁴ A L Q T P — — — — — — Y T F G Q rel. oomcaa⁵ 67%57% 62% 33% 80% 82% 100% 100% 100% 100% 100% 41% 100% 100% 100% 88% posoccupied⁶ 3  3  3  7  3  3  1  1  1  1  1  7  1  1  1  2 Framework IVamino acid¹ 101 102 103 104 105 106 A 107 108 Sum A 71 B  1 3 C 43 D 112E 13 71 F 72 G 16  1 233 H 26 I 14 94 K 12 13 66 L 11 219 M 37 N 56 P159 Q 159 R  4 12 126 S 325 T 16 140 V  5 146 W 31 X 3 Y 123 — 13 134unknown (?) 2 not sequenced 6  6  6  6  7  8  9  9 10 211 sum of seq² 1616 16 16 15 14 13 13 12 oomcaa³ 16 16 12 11 13 14 13 13 12 mcaa⁴ G T K LE I — K R rel. oomcaa⁵ 100% 100% 75% 69% 87% 100% 100% 100% 100% posoccupied⁶ 1  1  2  2  3  1  1  1  1

TABLE 4C Analysis of V kappa subgroup 3 Framework I amino acid¹ 1 2 3 45 6 7 8 9 10 11 12 13 14 15 16 A  5  2 27  1 B  1 C  2 D  2 14 E 76 27 F 1  1 G  1 82  1 152  H  1 I 75 K  3 L  4  1 104   1 150  129   1 M  513 N  5 P 124  147  Q 123  R  1 S 119   3  1 150   1 141  T  2 117  147  5  1 V  1 89  1  1  1 22  1 W X Y — unknown (?) not sequenced sum ofseq² 88 88 117  118  118  123  123  124  126  149  151  152  152  152 152  152  oomcaa³ 76 75 89 104  117  123  119  124  82 147  150  150 129  141  147  152  mcaa⁴ E I V L T Q S P G T L S L S P G rel. oomcaa⁵86% 85% 76% 88% 99% 100% 97% 100% 65% 99% 99% 99% 85% 93% 97% 100% posoccupied⁶  6  6  3  3  2  1  4  1  4  3  2  2  3  4  6  1 Framework ICDR I amino acid¹ 17 18 19 20 21 22 23 24 25 26 27 A B C D E A 178   2166   1 B C 181   1 D  6 E 146   1  1 F  7  1 G  1  1  1  1  1 H 17 I  1 5  2 K  1  5 L 173   1  1 M N  9 P Q 159  R 175  176   1  1 10 S 180  7 175  87 T  1 174   7  2  1 V  1  4  1  1  1 W  1 X Y  1  1 — 72 182 182  182  182  unknown (?)  1 not sequenced sum of seq² 153  181  182 182  182  182  181  182  182  181  181  182  182  182  182  182  oomcaa³146  175  178  174  173  180  181  176  166  175  159  87 182  182  182 182  mcaa⁴ E R A T L S C R A S Q S — — — — rel. oomcaa⁵ 95% 97% 98% 96%95% 99% 100% 97% 91% 97% 88% 48% 100% 100% 100% 100% pos occupied⁶  3  7 2  4  3  3  1  3  5  6  6  8  1  1  1  1 CDR I Framework II amino acid¹F 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 A  1  1 181  B C D  1  1 2  1 E  1  1  1 F  1  7  1 G  2  7  3  1  2  1 184  H  1  2  1 12  1  1I 24  4  1  1 K  1  1 153  L  8  1  1 176   3  2 M N  3 12 25 32 P  1170  Q  1  1 183  167   1 181  R 10  3 18 16  1  1 27  5 S 72 86 151 118   4  5 T  1  1  3  8  1  1 V 76 68  1  7  3  2 W  5 185  X Y  1  1115  183  — 182  unknown (?)  1 not sequenced sum of seq² 182  182  182 181  181  182  183  184  185  185  185  185  184  184  184  184  oomcaa³182  76 86 151  118  115  176  181  185  183  183  167  153  170  184 181  mcaa⁴ — V S S S Y L A W Y Q Q K P G Q rel. oomcaa⁵ 100% 42% 47% 83%65% 63% 96% 98% 100% 99% 99% 90% 83% 92% 100% 98% pos occupied⁶  1  6 1110 13 12  2  3  1  3  2  4  6  6  1  3 Framework Framework II CDR II IIIamino acid¹ 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 A 176   4147  176   1 B C  1 D 43  2  4 E F  1  1  4 G 125   2 10 179  H  9  1 I178   1 168  K  1  7  1 L  1 179  174   1 M  3  1 N  1  1 53  2 P  5184   2  2  2 Q  1 R 182   1  4 180  S  3  6  4 179  74  1  5 T  3 11  244 164   2 V  3  9  3 19  3 15 W  1  1 X Y 165   2 — unknown (?)  1 notsequenced sum of seq² 184  185  185  183  183  183  183  183  183  183 183  183  185  185  185  185  oomcaa³ 176  184  182  179  174  178  165 125  147  179  74 180  176  164  179  168  mcaa⁴ A P R L L I Y G A S S RA T G I rel. oomcaa⁵ 96% 99% 98% 98% 95% 97% 90% 68% 80% 98% 40% 98% 95%89% 97% 91% pos occupied⁶  3  2  3  3  2  4  6  7  6  3  6  4  5  7  3 3 Framework III amino acid¹ 59 60 61 62 63 64 65 66 67 68 69 70 71 7273 74 A 68  3  5  3  1  3 B C D 112   1 152  E  1  1 30 F 183  183   2 G184   3 178  — 177  H  1 I  1  1  3 K  1 L  1 182  M  1 N  1  1 P 177  Q 1 R 182   2  1  2 S  7 180  179  185   3  7  2 T  1  2  3  2 177  172 179  V  3  1  1 W  1 X Y  1 — unknown (?)  1 not sequenced sum of seq²185  185  185  185  185  185  185  185  185  185  185  184  184  184 184  184  oomcaa³ 177  112  182  183  180  184  179  178  185  177  177 152  183  172  182  179  mcaa⁴ P D R F S G S G S G T D F T L T rel.oomcaa⁵ 96% 61% 98% 99% 97% 99% 97% 96% 100% 96% 96% 83% 99% 93% 99% 97%pos occupied⁶  3  5  3  3  3  2  4  5  1  5  4  4  2  5  2  3 FrameworkIII CDR III amino acid¹ 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90A  3 174  B  1 C  2  1 182  D  1  3 182  E 149  175   2 F  1 178   2  1 4 G  3  1  2 H  1  1  7 I 178   1  1  9 K  1 L 178   1  1  7  1  1 M  1 5 N  1  5 P 149  Q 34  1 181  155  R  1 111   3  1 S 169  65 34  1  2 T 8  4  1  8 V  4  6  1  3 159   7 W X Y  1  1 183  176   1  2 — unknown(?) not sequenced sum of seq² 184  184  184  184  184  184  182  184 184  184  184  184  184  183  183  183  oomcaa³ 178  169  111  178  149 149  175  182  178  174  159  183  176  182  181  155  mcaa⁴ I S R L E PE D F A V Y Y C Q Q rel. oomcaa⁵ 97% 92% 60% 97% 81% 81% 96% 99% 97% 95%86% 99% 96% 99% 99% 85% pos occupied⁶  4  5  5  2  3  3  4  3  6  6  7 2  5  2  3  8 CDR III Framework IV amino acid¹ 91 92 93 94 95 A B C D EF 96 97 98 99 100 A  1  8  3  3  1 B C  2  1  2 D  8  5  1 E  2  1 F  5 2  7 166  G  1 104  15  1  1  2  1 166  41 H  4  1  2 I  1  1  4 K  2 1  1  1 L  2  7  5 42 M  1  1  2 N 28 71  1 P  1 139  24  7  2  9 Q  1 1  3  1  3 114 R 34  2  3  2  2 19 S  2 33 58 102  15  2  1  8 T  2 13 1  1  2  1 154  V  3  1  2 W 69 24 X Y 134   1  1 43 —  3  3  7 127 167  169  169  169  169   8  1  1  1  1 unknown (?) not sequenced 14 1414 14 14 14 14 17 16 16 16 sum of seq² 183  183  183  182  182  169 169  169  169  169  169  169  166  167  167  167  oomcaa³ 134  104  71102  139  127  167  169  169  169  169  43 154  166  166  114  mcaa⁴ Y GN S P — — — — — — Y T F G Q rel. oomcaa⁵ 73% 57% 39% 56% 76% 75% 99%100% 100% 100% 100% 25% 93% 99% 99% 68% pos occupied⁶  8 11 13  8 11 12 2  1  1  1  1 18  5  2  2  6 Framework IV amino acid¹ 101 102 103 104105 106 A 107 108 sum A 1345 B  2 C 375 D 23 564 E  3 141  759 F  6 765G 166   1 1804 H  1 64 I 143  803 K 152  157  489 L 54  1  2 1596 M  336 N  1  3 255 P  1  1 1147 Q  1  1 1314 R  9  2  4 134  1326 S  2 2629T 162   1  1 1593 V 111  11 646 W 287 X Y  1 1014 —  1  1  1  1  1  1166  1  1 2151 unknown (?)  4 not sequenced 16 16 15 16 16 16 17 17 45337 sum of seq² 167  167  168  167  167  167  166  166  138  oomcaa³166  162  152  111  141  143  166  157  134  mcaa⁴ G T K V E I — K Rrel. oomcaa⁵ 99% 97% 90% 66% 84% 86% 100% 95% 97% pos occupied⁶  2  5  7 4  5  7  1  5  4

TABLE 4D Analysis of V kappa subgroup 4 Framework I amino acid¹ 1 2 3 45 6 7 8 9 10 11 12 13 14 15 16 17 18 A 24  1 B C  1  1 D 25 26 E 25 F G 1 24 H I 26 K  1 L  1 26 26 M 24 N  1 P 26  1 Q  1 25 R 26 S 26 25 26 1 T 26 V 25  1 26 W X Y — unknown (?) not sequenced  7  7  7  7  7  7 7  7  7  7  7  7  7  7  7  7  7  7 sum of seq² 26 26 26 26 26 26 26 2626 26 26 26 26 26 26 26 26 26 oomcaa³ 25 26 25 24 26 25 26 26 26 25 2624 26 26 26 24 25 26 mcaa⁴ D I V M T Q S P D S L A V S L G E R rel.oomcaa⁵ 96% 100% 96% 92% 100% 96% 100% 100% 100% 96% 100% 92% 100% 100%100% 92% 96% 100% pos occupied⁶  2  1  2  3  1  2  1  1  1  2  1  3  1 1  1  3  2  1 Framework I CDR I amino acid¹ 19 20 21 22 23 24 25 26 27A B C D E F 28 29 30 A 26  1  1 B C 33 D  1  1  1 E F G H I 26  1 K 33 2 30 L  2 31 M N 26 30 31  1 P  1  1 Q 32  1 R  1  1  1 S 31 33 33 3232  1 T 26  1 V 28  2 W X Y 32 — unknown (?) not sequenced  7  7  7  7sum of seq² 26 26 26 26 33 33 33 33 33 33 33 33 33 33 33 33 33 33oomcaa³ 26 26 26 26 33 33 31 33 32 33 28 31 32 32 32 30 31 30 mcaa⁴ A TI N C K S S Q S V L Y S S N N K rel. oomcaa⁵ 100% 100% 100% 100% 100%100% 94% 100% 97% 100% 85% 94% 97% 97% 97% 91% 94% 91% pos occupied⁶  1 1  1  1  1  1  3  1  2  1  5  2  2  2  2  3  3  4 CDR I Framework IIamino acid¹ 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 A 32 2 B C D E  1 F G 32 H  2 I 32 K 33 32 L 33 29 33 M  1 N 33 P 31 31 33 Q32 33 32 R  1  1  1 S  2 T  1 V  4 W 33 X Y 33 31 — unknown (?) notsequenced sum of seq² 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 33 3333 oomcaa³ 33 33 33 32 33 31 32 33 33 31 32 32 31 33 32 29 33 32 mcaa⁴ NY L A W Y Q Q K P G Q P P K L L I rel. oomcaa⁵ 100% 100% 100% 97% 100%94% 97% 100% 100% 94% 97% 97% 94% 100% 97% 88% 100% 97% pos occupied⁶  1 1  1  2  1  2  2  1  1  2  2  2  2  1  2  2  1  2 Frame- work II CDR IIFramework III amino acid¹ 49 50 51 52 53 54 55 56 57 58 59 60 61 62 6364 65 66 A 30 B C D 33 E 32 F 33 G 33  1 33 33 H I  1 K L M N  2 P  1 33 1 Q R 33 32 S  1 31  1 33 32 33 T  2  1 29 V  1 33 W 33 X Y 33 —unknown (?) not sequenced sum of seq² 33 33 33 33 33 33 33 33 33 33 3333 33 33 33 33 33 33 oomcaa³ 33 33 30 31 29 33 32 33 33 33 33 33 32 3332 33 33 33 mcaa⁴ Y W A S T R E S G V P D R F S G S G rel. oomcaa⁵ 100%100% 91% 94% 88% 100% 97% 100% 100% 100% 100% 100% 97% 100% 97% 100%100% 100% pos occupied⁶  1  1  3  3  4  1  2  1  1  1  1  1  2  1  2  1 1  1 Framework III amino acid¹ 67 68 69 70 71 72 73 74 75 76 77 78 7980 81 82 83 84 A 33 32 B C D 32 33 E 33 F 32 G 33  1  1 H I 33 K L 33 32M  1 N  2  1 P Q 32 R  1 S 33 30 32 T 33 33 33  1 V  1 33 W X Y —unknown (?) not sequenced sum of seq² 33 33 33 33 33 33 33 33 33 33 3333 33 33 33 33 33 33 oomcaa³ 33 33 33 32 32 33 33 33 33 30 32 32 32 3333 33 33 32 mcaa⁴ S G T D F T L T I S S L Q A E D V A rel. oomcaa⁵ 100%100% 100% 97% 97% 100% 100% 100% 100% 91% 97% 97% 97% 100% 100% 100%100% 97% pos occupied⁶  1  1  1  2  2  1  1  1  1  3  2  2  2  1  1  1 1  2 Framework III CDR III amino acid¹ 85 86 87 88 89 90 91 92 93 94 95A B C D E F 96 A  1 B C 33 D  1  1 E F  1  1 G  2 H  1  3 I  2 K L  1  2 1  3  1 M N  4  4 P  1 29  1  4 Q 30 32  1  1 R  1  1  2 S  2 23  2  1T  2 22 V 33 W  2 X Y 33 31 31 29  1 — 13 15 15 15 15 15  3 unknown (?)not sequenced 18 18 18 18 18 18 18 sum of seq² 33 33 33 33 33 33 33 3333 33 33 15 15 15 15 15 15 15 oomcaa³ 33 33 31 33 30 32 31 29 23 22 2913 15 15 15 15 15  4 mcaa⁴ V Y Y C Q Q Y Y S T P — — — — — — P rel.oomcaa⁵ 100% 100% 94% 100% 91% 97% 94% 88% 70% 67% 88% 87% 100% 100%100% 100% 100% 27% pos occupied⁶  1  1  3  1  2  2  2  4  6  7  3  3  1 1  1  1  1  8 CDR III Framework IV amino acid¹ 97 98 99 100 101 102 103104 105 106 A 107 108 sum A 183 B C 68 D 154 E 14 105 F 15 82 G 15  4 15228 H  6 I 14 135 K 14 13 158 L  4 258 M  1 27 N  1 136 P  1 195 Q 11  1264 R  1  1  1 11 116 S  2  1 499 T 12 14 236 V  9 196 W  1 69 X Y 254 —15 106 unknown (?) not sequenced 18 18 18 18 18 18 18 18 18 18 18 18 22518 sum of seq² 15 15 15 15 15 15 15 15 15 15 15 15 11 oomcaa³ 12 15 1511 15 14 14  9 14 14 15 13 11 mcaa⁴ T F G Q G T K V E I — K R rel.oomcaa⁵ 80% 100% 100% 73% 100% 93% 93% 60% 93% 93% 100% 87% 100% posoccupied⁶  3  1  1  2  1  2  2  4  2  2  1  3  1

TABLE 5A Analysis of V lambda subgroup 1 Framework I amino acid¹ 1 2 3 45 6 7 8 9 10 11 12 13 14 15 A 19 18 20 B C D E F G 22 H  2 I  1  1 K L 1 41  1 M N P 41 41  1 41 Q 22  1 41 R S 39 41 41  1 T 41 19 V  1 38 20 1  1 W X Y Z 16 — 41 unknown (?) not sequenced  2  2  1  1  1  1  1  1 1  1  1  1  1  1 sum of seq² 40 40 41 41 41 41 41 41 41 41 41 41 41 4142 oomcaa³ 22 39 38 41 41 41 41 41 41 41 20 41 22 20 41 mcaa⁴ Q S V L TQ P P S — V S G A P rel. oomcaa⁵ 55% 98% 93% 100% 100% 100% 100% 100%100% 100% 49% 100% 54% 49% 98% pos occupied⁶  3  2  4  1  1  1  1  1  1 1  4  1  3  4 2 Framework I CDRI amino acid¹ 16 17 18 19 20 21 22 23 2425 26 27 D E 28 A  2  1 B C 42 D  3 E  1 F  1  1 G 42 42  3  1  2 H I  141  1 37 K 14  1 L  1  1 M  1 N  2  1 37 P Q 42 R 25  1  1 S  1  1 42 3834 34 38 T  1 38  3  4  3  2 V 42  1 W X Y Z — unknown (?) not sequencedsum of seq² 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 oomcaa³ 42 4225 42 38 41 42 42 38 42 34 34 38 37 37 mcaa⁴ G Q R V T I S C S G S S S NI rel. oomcaa⁵ 100% 100% 60% 100% 90% 98% 100% 100% 90% 100% 81% 81% 90%88% 88% pos occupied⁶  1  1  5  1  4  2  1  1  3  1  4  6  4  4  5 CDRIFramework II amino acid¹ 29 30 31 A 32 33 34 35 36 37 38 39 40 41 42 A 2  2  1  4 B C D  3  1  3  1  1 E  1 F  1  1  1  4 G 39  4  2 39 H  2 2  2  1  1  6  1 I  1 K  1  1 L  1 31 M  1 N 13 31  2  1  9 P  1 42  1Q  1 39 34 R  5  2  1  1 S 13  1  1  3 19 T  1  1  7  2 36 V  2 40  1  5W 42 X Y  4  1 20  7 40 Z — 36 unknown (?) not sequenced  1  1  1  1 sumof seq² 42 42 42 41 41 41 41 42 42 42 42 42 42 42 42 oomcaa³ 39 13 31 3620 40 19 42 40 39 34 31 42 39 36 mcaa⁴ G N N — Y V S W Y Q Q L P G Trel. oomcaa⁵ 93% 31% 74% 88% 49% 98% 46% 100% 95% 93% 81% 74% 100% 93%86% pos occupied⁶  3  8  7  5 10  2  7  1  3  3  4  5  1  4  4 FrameworkII CDR II amino acid¹ 43 44 45 46 47 48 49 50 51 52 53 54 55 56 A A 40 1  1 B C D 13 10  8 E  2  5  1 F  1 G  1 H  1  1 I 40  1 K 35  1  1 18L 41 40  1  1  1 M  1  1 N  1  3 28 30  2 P 42 38 Q 15 R  4  7  2 40 S 1  9  2  3  1  2 40 T  1  1 V  1  2  1 W  1 X Y 40  1  1 Z — 41 unknown(?) not sequenced  1  1 sum of seq² 42 42 42 42 42 42 42 42 42 42 42 4241 41 41 oomcaa³ 40 42 35 41 40 40 40 13 28 30 18 40 38 40 41 mcaa⁴ A PK L L I Y D N N K R P S — rel. oomcaa⁵ 95% 100% 83% 98% 95% 95% 95% 31%67% 71% 43% 95% 93% 98% 100% pos occupied⁶  3  1  4  2  2  3  3 10  5  4 9  3  3  2  1 CDR II Framework III amino acid¹ B C D E 57 58 59 60 6162 63 64 65 66 A A  5 B C D 38 E F 38 G 41  2 36 H  1 I 17  3 K 38 L  1M N P 38 Q R 42  4 S  2 42 42 T  1 V 24  1 W X Y Z — 41 41 41 42 42unknown (?) not sequenced  1  1  1  1 sum of seq² 41 41 41 42 41 41 4141 42 42 42 42 42 42 42 oomcaa³ 41 41 41 42 41 24 38 38 42 38 42 36 4238 42 mcaa⁴ — — — — G V P D R F S G S K — rel. oomcaa⁵ 100% 100% 100%100% 100% 59% 93% 93% 100% 90% 100% 86% 100% 90% 100% pos occupied⁶  1 1  1  1  1  2  3  3  1  3  1  3  1  2  1 Framework III amino acid¹ B 6768 69 70 71 72 73 74 75 76 77 78 79 80 A  1  3 41 24  2 B C D  1 E  1 FG 40 17  1 42 H  1 I 41 K L 42 41 M N P  2 Q 31 R  8 S 42  1 42 24 20 20T 38 18 21 17 V  1  1  1  1  1 W  1 X Y Z — 42 unknown (?) not sequencedsum of seq² 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 oomcaa³ 42 4240 38 42 41 24 42 24 41 21 42 41 31 20 mcaa⁴ — S G T S A S L A I T G L QS rel. oomcaa⁵ 100% 100% 95% 90% 100% 98% 57% 100% 57% 98% 50% 100% 98%74% 48% pos occupicd⁶  1  1  3  3  1  2  2  1  3  2  3  1  2  5  5Framework III CDR III amino acid¹ 81 82 83 84 85 86 87 88 89 90 91 92 9394 95 A 38  1 22 15  1 B C 42 D  1 41 37 39 17 E 24 42  1 F  2  1 G 1514  1 H  2  1 I  1 K L  1 37 M N  1  2  2 P  1 Q  3 R  5  1 S  1  4 1735 T  3 22  1  1 V  1  1  1 W  2 38 X Y 42 39  3  1 Z — unknown (?) notsequenced  1  1  1  1  1  1  1 sum of seq² 42 42 42 42 42 42 42 42 41 4141 41 41 41 41 oomcaa³ 24 41 42 38 37 42 39 42 22 22 38 39 17 35 37mcaa⁴ E D E A D Y Y C A T W D D S L rel. oomcaa⁵ 57% 98% 100% 90% 88%100% 93% 100% 54% 54% 93% 95% 41% 85% 90% pos occupied⁶  4  2  1  3  5 1  3  1  5  3  2  2  8  3  5 CDR III Framework IV amino acid¹ A B C D EF 96 97 98 99 100 101 102 103 104 A 16  4  1 B C D  7 E  1  1  1 F 36 G17  1  5  1 36 31 36 H  1 I  1  1 K  1 30 L  1  1 25 M  1 N  9  1  1 P 6 Q  3 R  2  2  1 S 18  1  1  1 T  1  3 36  1 V  2  9 34 11 W  7 X Y  3Z —  2  4 35 39 38 38  1 unknown (?) not sequenced  1  1  3  3  3  3  3 3  4  4  6  6  6  6  6 sum of seq² 41 41 39 39 38 38 39 39 36 36 36 3636 36 36 oomcaa³ 18 17 35 39 38 38  9 34 36 36 31 36 36 30 25 mcaa⁴ S G— — — — V V F G G G T K L rel. oomcaa⁵ 44% 41% 90% 100% 100% 100% 23%87% 100% 100% 86% 100% 100% 83% 69% pos occupied⁶  8  6  5  1  1  1 10 6  1  1  4  1  1  5  2 Framework IV amino acid¹ 105 106 A 107 108 sum A285 B C 84 D 224 E 81 F 87 G 26 559 H 25 I 188 K 141 L 34 344 M  5 N 176P  1 296 Q  1 18 251 R  2 156 S  2 720 T 36 359 V 36  1 282 W  1 92 X Y202 Z 16 — 524 unknown (?) not sequenced  6  6  6 10 22 141 sum of seq²36 36 36 31 19 oomcaa³ 36 36 34 26 18 mcaa⁴ T V L G Q rel. oomcaa⁵ 100%100% 94% 84% 95% pos occupied⁶  1  1  3  4  2

TABLE 5B Analysis of V lambda subgroup 2 Framework I amino acid¹ 1 2 3 45 6 7 8 9 10 11 12 13 14 15 A 35 30  6  1  1 B C D E F G 42 H  2 I  1 KL 40  3 M N P 42  6 40 Q 22  4 41 R  6  1 S 41 40 42 42 T 42  1 V  1  236 W X Y Z 16 — 42 unknown (?)  1 not sequenced  3  1  1  3  1  1  1  1 1  1  1  1 sum of seq² 40 42 42 40 42 42 42 42 42 42 42 42 43 43 43oomcaa³ 22 41 35 40 42 41 42 30 40 42 36 42 42 42 40 mcaa⁴ Q S A L T Q PA S — V S G S P rel. oomcaa⁵ 55% 98% 83% 100% 100% 98% 100% 71% 95% 100%86% 100% 98% 98% 93% pos occupied⁶  3  2  4  1  1  1  1  3  3  1  2  1 2  2  2 Framework I CDRI amino acid¹ 16 17 18 19 20 21 22 23 24 25 2627 D E 28 A  3  1 B C 42  1 D  1 39 E F  1 G 42 43  1 H  1  1 I 28 41  1 6 K L  1  1 M N  1  3  4 P  1 Q 42 R  1 S 43 42  3  3 35 38 T 43 36 39 3 V 14 37 W X Y  1 Z — unknown (?) not sequenced  1  1 sum of seq² 4343 43 43 43 43 42 42 43 43 43 43 43 43 43 oomcaa³ 42 42 43 28 43 41 4242 36 43 39 35 38 39 37 mcaa⁴ G Q S I T I S C T G T S S D V rel. oomcaa⁵98% 98% 100% 65% 100% 95% 100% 100% 84% 100% 91% 81% 88% 91% 86% posoccupied⁶  2  2  1  3  1  3  1  1  4  1  3  7  4  2  2 CDRI Framework IIamino acid¹ 29 30 31 A 32 33 34 35 36 37 38 39 40 41 42 A  1  1  1  4 BC  1 D  1  4  5  1  2 E  1 F  1  4  2 G 39 26 36 H  1  1  2 34 I  1 K  440 L  4  1  1 M N  1  4  3 28  2 P 41 Q 41 39 R  2  1  1 S  5  1  2  4 1 42  1 T  1  1  1 V 41  1 W 43 X Y  1 37 29 41  5 Z —  1 unknown (?) 1  1  1 not sequenced  1  1 sum of seq² 43 43 43 43 43 42 42 43 43 4343 43 43 43 43 oomcaa³ 39 26 37 28 29 41 42 43 41 41 39 34 41 36 40mcaa⁴ G G Y N Y V S W Y Q Q H P G K rel. oomcaa⁵ 91% 60% 86% 65% 67% 98%100% 100% 95% 95% 91% 79% 95% 84% 93% pos occupied⁶  5  7  5  7  6  2  1 1  2  2  3  5  3  4  4 Framework II CDR II amino acid¹ 43 44 45 46 4748 49 50 51 52 53 54 55 56 A A 40 B C D 20  1  2  1 E 20  2 F  7  1 G  2 2  1 H  1 I  1  9 43  1 K 41  1 21 L 38  6 M 26  1 N  1  8 12 P 43 43 Q 2 R  2 43 S  2 21  3 43 T  7 V  3  4  2 39 W X Y 34  2 Z — 43 unknown(?) not sequenced sum of seq² 43 43 43 43 43 43 43 43 43 43 43 43 43 4343 oomcaa³ 40 43 41 38 26 43 34 20 39 21 21 43 43 43 43 mcaa⁴ A P K L MI Y D V S K R P S — rel. oomcaa⁵ 93% 100% 95% 88% 60% 100% 79% 47% 91%49% 49% 100% 100% 100% 100% pos occupied⁶  2  1  2  3  4  1  3  4  4  8 8  1  1  1  1 CDR II Framework III amino acid¹ B C D E 57 58 59 60 6162 63 64 65 66 A A  2 B C  1 D 17 E F 42 G 43  1 41 H  2 I  3 K 42 L  1 1 M N 19 P 15 Q R 43  1 S 28  2 43 42 T V 39 W X Y  2 Z — 43 43 43 4343 unknown (?) not sequenced sum of seq² 43 43 43 43 43 43 43 43 43 4343 43 43 43 43 oomcaa³ 43 43 43 43 43 39 28 19 43 42 43 41 42 42 43mcaa⁴ — — — — G V S N R F S G S K — rel. oomcaa⁵ 100% 100% 100% 100%100% 91% 65% 44% 100% 98% 100% 95% 98% 98% 100% pos occupied⁶  1  1  1 1  1  3  2  6  1  2  1  2  2  2  1 Framework III amino acid¹ B 67 68 6970 71 72 73 74 75 76 77 78 79 80 A  3  1 43 36 B C D  1  2 E  1 F G 3942 H I 35 K  1 L 43 43 M N 38 P  2 Q 41 R  2 S 42  1 43 42 T  1 41 43  1 2 V  8  3 W X Y Z — 43 unknown (?)  1 not sequenced  1 sum of seq² 4342 43 43 43 43 43 43 43 43 43 43 43 43 43 oomcaa³ 43 42 39 38 41 43 4343 43 35 42 42 43 41 36 mcaa⁴ — S G N T A S L T I S G L Q A rel. oomcaa⁵100% 100% 91% 88% 95% 100% 100% 100% 100% 81% 98% 98% 100% 95% 84% posoccupied⁶  1  1  3  4  3  1  1  1  1  2  2  2  1  2  4 Framework III CDRIII amino acid¹ 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 A 43  2  121  1 B C 43 11 D  3 42 39  3  1  2 E 38 43  1  1 F  3  3  1 G  1  1 21 3  4 H  2  1 I  1  1  1 K  3 L M N  1  1  1  5  7 P  1 Q  1 R  2  3 S 1 30 41 12 23 14 T 16  4  4  3 V  1 W X Y 43 39 39  1  6 Z —  1 unknown(?)  1  2 not sequenced  1 sum of seq² 43 43 43 43 43 43 43 43 43 42 4343 43 43 43 oomcaa³ 38 42 43 43 39 43 39 43 30 41 39 21 21 23 14 mcaa⁴ ED E A D Y Y C S S Y A G S S rel. oomcaa⁵ 88% 98% 100% 100% 91% 100% 91%100% 70% 98% 91% 49% 49% 53% 33% pos occupied⁶  4  2  1  1  3  1  3  1 3  2  3  7  7  8 11 CDR III Framework IV amino acid¹ A B C D E F 96 9798 99 100 101 102 103 104 A  1  1  1 B C D  1 E F  1  5 42 G  1 42 33 42H I  2  1  7 K 36 L  1  1  6  5 28 M  1  1 N  5  1  1 P  4 Q  2  1 R  1 5  1  2 S  9  1 T 21  7 41 V 11 28 14 W  5 X Y  4  1 Z —  3 36 42 43 4343 unknown (?) not sequenced  1  1  1  1  1  1  2  2  1 sum of seq² 4243 43 43 43 43 43 42 42 42 42 42 41 41 42 oomcaa³ 21 36 42 43 43 43 1128 42 42 33 42 41 36 28 mcaa⁴ T — — — — — V V F G G G T K L rel. oomcaa⁵50% 84% 98% 100% 100% 100% 26% 67% 100% 100% 79% 100% 100% 88% 67% posoccupied⁶  6  5  2  1  1  1 13  5  1  1  4  1  1  5  2 Framework IVamino acid¹ 105 106 A 107 108 sum A 280 B C 99 D 188 E 107 F 113 G 19567 H 48 I  1 184 K 189 L 40 264 M 29 N 146 P 238 Q 14 250 R  4 121 S  1 2 831 T 40 398 V 42  1 327 W 48 X Y 285 Z 16 — 555 unknown (?)  8 notsequenced  1  1  2 15 28 80 sum of seq² 42 42 41 25 14 oomcaa³ 40 42 4019 14 mcaa⁴ T V L G Q rel. oomcaa⁵ 95% 100% 98% 76% 100% pos occupied⁶ 3  1  2  3  1

TABLE 5C Analysis of V lambda subgroup 3 Framework I amino acid¹ 1 2 3 45 6 7 8 9 10 11 12 13 14 15 A  1  1  2  7 20  1 B C D  5 10 E 20  1 F  1 1  1  1 G  1 H I K L 37  4  1  9 M N P 26 35  1 27 Q  4  4 38 R S 13 14 1  1 28 37 18 T 36  1 V  8  1  2 34 36 W X Y 23 Z — 20 38 unknown (?)not sequenced sum of seq² 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38oomcaa³ 20 23 20 37 36 38 26 35 28 38 34 37 36 20 27 mcaa⁴ — Y E L T Q PP S — V S V A P rel. oomcaa⁵ 53% 61% 53% 97% 95% 100% 68% 92% 74% 100%89% 97% 95% 53% 71% pos occupied⁶  4  3  5  2  3  1  4  3  4  1  2  2  3 2  4 Framework I CDRI amino acid¹ 16 17 18 19 20 21 22 23 24 25 26 27 DE 28 A 27  1  5 B C 38 D 30  1 E  1  2  2 F G 37  9 38  1 H  1 I 38  9 K 2  7 L 28 M  1 N  2  4  9  1 P  1  1 Q 36 10 R 25  2 S  9  1 19 10 T 38 3 33  1 V 10 W X Y  1 Z — 38 38 unknown (?) not sequenced sum of seq²38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 oomcaa³ 37 36 38 27 25 3833 38 19 38 30 10 38 38 28 mcaa⁴ G Q T A R I T C S G D S — — L rel.oomcaa⁵ 97% 95% 100% 71% 66% 100% 87% 100% 50% 100% 79% 26% 100% 100%74% pos occupied⁶  2  2  1  3  4  1  5  1  3  1  5  9  1  1  3 CDRIFramework II amino acid¹ 29 30 31 A 32 33 34 35 36 37 38 39 40 41 42 A 1  1 21  3 B C  5 D 10  3  1 E  1  3  6  1 F  1  2  3 G 23  4 36 H  2 9  1 I  1 K  2 13 32 L  2 M  1 N  2  1  2 P  3 36  1 Q  4 37 35  1 36 R10  1  1  1  4  2 S 11  2  8 14  1  2 T  1  4 V  1 15 W 38 X Y  8 20  1 4 35 Z — 37 unknown (?) not sequenced  1  1 sum of seq² 38 38 37 37 3738 38 38 38 38 38 38 38 38 38 oomcaa³ 23 11 13 37 20 21 14 38 35 37 3532 36 36 36 mcaa⁴ G S K — Y A S W Y Q Q K P G Q rel. oomcaa⁵ 61% 29% 35%100% 54% 55% 37% 100% 92% 97% 92% 84% 95% 95% 95% pos occupied⁶  5  9  9 1  7  4  7  1  2  2  3  4  2  2  3 Framework II CDR II amino acid¹ 4344 45 46 47 48 49 50 51 52 53 54 55 56 A A 23  1  1  1 B C D  9 22  2  8E  5  3  3 F  2  1 G  9  2 — H  1  3  1 I  1 28  1 K  2  6  1 13 L  6 33 1 M  1  1 N  1 19  9 P 38 37  1 Q  9  1 R  1  1  1 38 S 14 10  1  1 36T  2  4 V  1 31  4 37  9 W X Y 35 Z — 38 unknown (?) not sequenced sumof seq² 38 38 38 38 38 38 38 38 38 38 38 38 38 38 38 oomcaa³ 23 38 31 3337 28 35  9 22 19 13 38 37 36 38 mcaa⁴ A P V L V I Y D D N K R P S —rel. oomcaa⁵ 61% 100% 82% 87% 97% 74% 92% 24% 58% 50% 34% 100% 97% 95%100% pos occupied⁶ 3  1  3  3  2  3  3  7  8  7  9  1  2  3  1 CDR IIFramework III amino acid¹ B C D E 57 58 59 60 61 62 63 64 65 66 A A B CD  9 E 27 F 38 G 38 38 H I 37 K L M N 21 P 36 Q R 38 S  1 38 38 12 T  5V W X Y Z — 38 38 38 38 38 unknown (?)  1 not sequenced  1  1  1 sum ofseq² 38 38 38 38 38 37 37 37 38 38 38 38 38 38 38 oomcaa³ 38 38 38 38 3837 36 27 38 38 38 38 38 21 38 mcaa⁴ — — — — G I P E R F S G S N — rel.oomcaa⁵ 100% 100% 100% 100% 100% 100% 97% 73% 100% 100% 100% 100% 100%55% 100% pos occupied⁶ 1  1  1  1  1  1  2  2  1  1  1  1  1  3  1Framework III amino acid¹ B 67 68 69 70 71 72 73 74 75 76 77 78 79 80 A 1 36  1  1 11  1 34 B C D E 10 F G 37 28 H  1 I  1  1 37  1 K  1 L 38 MN 28  1 P Q  1 25 R  1 10  1 S 37  2 11 23  1 T  1  6 37 25 36 12 13  2V  2  1 14  1  1 W X Y Z — 38 unknown (?) not sequenced sum of seq² 3838 38 38 38 38 38 38 38 38 38 38 38 38 38 oomcaa³ 38 37 37 28 37 36 2538 36 37 23 28 14 25 34 mcaa⁴ — S G N T A T L T I S G V Q A rel. oomcaa⁵100% 97% 97% 74% 97% 95% 66% 100% 95% 97% 61% 74% 37% 66% 89% posoccupied⁶  1  2  2  5  2  2  4  1  3  2  5  2  3  5  4 framework III CDRIII amino acid¹ 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 A 38 13  3 2  1 B C 38 D 38 37 32  1  1 E 14 38  1  1 F  2  2 G 10  3 14 H I  1 KL  2  1  1  1 M 10  1 N 10  2  1  2 P  1 Q 25  1 R 10  1  2 S  1 14  128 26 13 T  1  3  7 V  1 11 W 23 X Y 38 36  1  1 Z — unknown (?) notsequenced  1  1  1  1 sum of seq² 38 38 38 38 38 38 38 38 38 38 38 37 3737 37 oomcaa³ 14 38 38 38 37 38 36 38 25 14 23 32 28 26 14 mcaa⁴ E D E AD Y Y C Q S W D S S G rel. oomcaa⁵ 37% 100% 100% 100% 97% 100% 95% 100%66% 37% 61% 86% 76% 70% 38% pos occupied⁶  6  1  1  1  2  1  2  1  5  3 5  4  7  8  6 CDR III Framework IV amino acid¹ A B C D E F 96 97 98 99100 101 102 103 104 A  2  4 B C D  6 E  2  2  2 F 35 G  3  1  3  1 35 3135 H 12  1 I  4 K  1 30 L  1  1  4  2 28 M  1  1 N 10  1 P  3  1 Q  1 R 2  2 S  1  1 T  2  4 35 V 18 28  7 W  1 X Y  1  3  1  3 Z — 10 15 31 3637 36  1 unknown (?) not sequenced  2  1  1  1  1  1  1  1  3  3  3  3 3  4  3 sum of seq² 36 37 37 37 37 37 37 37 35 35 35 35 35 34 35oomcaa³ 10 15 31 36 37 36 18 28 35 35 31 35 35 30 28 mcaa⁴ N — — — — — VV F G G G T K L rel. oomcaa⁵ 28% 41% 84% 97% 100% 97% 49% 76% 100% 100%89% 100% 100% 88% 80% pos occupied⁶  9  8  5  2  1  2  9  6  1  1  2  1 1  3  2 Framework IV amino acid¹ 105 106 A 107 108 sum A 265 B C  1 82D 225 E 145 F 90 G 24 461 H 32 I 160 K 110 L 33 233 M 17 N 126 P  1 249Q  7 275 R 154 S  2 501 T 35 347 V 35 308 W 62 X Y 211 Z — 603 unknown(?)  1 not sequenced  3  3  4 11 28 89 sum of seq² 35 35 34 27  7oomcaa³ 35 35 33 24  7 mcaa⁴ T V L G Q rel. oomcaa⁵ 100% 100% 97% 89%100% pos occupied⁶ 1  1  2  3  1

TABLE 6A Analysis of V heavy chain subgroup 1A Framework I amino acid¹ 12 3 4 5 6 7 8 9 10 11 12 13 14 15 A  1 14 60 B C D E  1  2  1  2 64 F G58  1 64 H  2 I  2 K  2 57 64 L  2 59  3 M  1 N  6 P 63 Q 53 56  2 45 R 1 S 60  3  1 T V  2 55  1 55 61 W X Y Z  3 — unknown (?) not sequenced11 10 10 10 10 10 10 10  6  6  6  6  6  6  6 sum of seq² 59 60 60 60 6060 60 60 64 64 64 64 64 64 64 oomcaa³ 53 55 56 59 55 45 60 58 60 64 6157 64 63 64 mcaa⁴ Q V Q L V Q S G A E V K K P G rel. oomcaa⁵ 90% 92% 93%98% 92% 75% 100% 97% 94% 100% 95% 89% 100% 98% 100% pos occupied⁶  4  4 3  2  4  3  1  2  3  1  2  3  1  2 1 Framework I amino acid¹ 16 17 1819 20 21 22 23 24 25 26 27 28 29 30 A 24  1 62  1 B C 63 D  1 E F 69 G 1 69 41  1 H  1 I  1 K 60 63  1 L M N  2 P Q R  3  1  1  1 S 40 63 6368  1 40 T  1  1  2 68 25 V 64 64 W X Y 27 Z — unknown (?) not sequenced 6  6  6  6  6  6  6  6  5  2  1 sum of seq² 64 64 64 64 64 64 64 64 6568 69 70 70 70 70 oomcaa³ 40 63 64 60 64 63 63 63 62 68 69 41 68 69 40mcaa⁴ S S V K V S C K A S G G T F S rel. oomcaa⁵ 63% 98% 100% 94% 100%98% 98% 98% 95% 100% 100% 59% 97% 99% 57% pos occupied⁶  2  2  1  3  1 2  2  2  3  1  1  4  3  2 6 CDR I Framework II amino acid¹ 31 A B 32 3334 35 36 37 38 39 40 41 42 43 A 41 70 B C D E F  3  3 G 23  1 68 H  1  1 1 I 61  1  1 K  1 L  1  2  1 M  4 N  5  4 P  1 68 Q 69 69 R  1 70  1  1S 60  2 60  1 T  3  3  4 V  1 69 W 70 X Y 64 Z — 70 70 unknown (?) notsequenced sum of seq² 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70oomcaa³ 60 70 70 64 41 61 60 70 69 70 69 70 68 68 69 mcaa⁴ S — — Y A I SW V R Q A P G Q rel. oomcaa⁵ 86% 100% 100% 91% 59% 87% 86% 100% 99% 100%99% 100% 97% 97% 99% pos occupied⁶  5  1  1  4  6  4  5  1  2  1  2  1 3  3 2 Framework II CDR II amino acid¹ 44 45 46 47 48 49 50 51 52 A B C53 54 55 A  1  5 B C D  1 E 69 F  2  3 39 G 69  1 69 39  1 68 H I 65 3834 K L 68  1  1  2  4 M 67  2  4 N  4  3 22 P  1 44 Q  1  1  1 R  1  4 1 S  1  1 22  1  1 T  1  2  4  1  3 V  1  2  2 16  1 W  1 67 26 X Y  120 Z — 70 70 unknown (?) not sequenced sum of seq² 70 70 70 70 70 70 7070 70 70 70 70 70 70 70 oomcaa³ 69 68 69 67 67 69 39 65 38 44 70 70 3439 68 mcaa⁴ G L E W M G G I I P — — I F G rel. oomcaa⁵ 99% 97% 99% 96%96% 99% 56% 93% 54% 63% 100% 100% 49% 56% 97% pos occupied⁶  2  3  2  4 4  2  4  4  6  5  1  1 10  6 3 CDR II Framework III amino acid¹ 56 5758 59 60 61 62 63 64 65 66 67 68 69 70 A  1 34 69 B C D 15  1  2 E  1 F 1 48  3  4 G  1  3 67 H  1 I  4  1 44 K  1  2  1 47  1  1 L  1  1 22  2 1 M 21 N  9 59 18 P  1  7 Q  1  1 70 64 R  2  2  1 69 S  1  2  1  5 T34 26  4  3 66 65 V  1 65  3 W X Y  1 68 Z — unknown (?) not sequencedsum of seq² 70 70 70 70 70 70 70 70 70 70 70 70 70 70 70 oomcaa³ 34 3459 68 69 70 47 48 64 67 69 65 66 44 65 mcaa⁴ T A N Y A Q K F Q G R V T IT rel. oomcaa⁵ 49% 49% 84% 97% 99% 100% 67% 69% 91% 96% 99% 93% 94% 63%93% pos occupied⁶ 11  6  7  3  2  1  4  2  5  3  2  3  3  4 2 FrameworkIII amino acid¹ 71 72 73 74 75 76 77 78 79 80 81 82 A B C A 43 64  1 B CD 70  2 E 33 64 F G  1 H  1  1 I  1  1  3  1  1 K  8 L  3  3 63 70 M 67N  4  1 16 P Q  1  3 R  1  3 23  1 S 70 62  1 41 49 T 24 27 67  1 69  2 3  2 V  3  3  4 W X Y 68 Z — unknown (?) not sequenced sum of seq² 7070 70 70 70 70 70 70 70 70 70 70 70 70 70 oomcaa³ 43 70 33 70 67 62 6964 68 67 64 63 41 49 70 mcaa⁴ A D E S T S T A Y M E L S S L rel. oomcaa⁵61% 100% 47% 100% 96% 89% 99% 91% 97% 96% 91% 90% 59% 70% 100% posoccupied⁶  3  1  5  1  2  4  2  4  3  2  4  3  6  6 1 Framework III CDRIII amino acid¹ 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 A  3  1 7066  2 16  1 B C 70  1 D 26 70 16  5  3 E 44  9 F  1  1  2  1 G  2 14 1320 H I  2  2  5 K  3  5  2 L  2  1  4  4  2 M  1  1  1  2 N  2  2 P 20 3 Q  1 R 62 55  1  5  7 S 67  1  1  1  5  5 T  4 67  1  3  3  5  4 V  164  3  3  2  4 W  1  1 X Y 69 68  1  2  3 Z —  1  2 unknown (?) notsequenced  2  2  2 sum of seq² 70 70 70 70 70 70 70 70 70 70 70 70 68 6868 oomcaa³ 62 67 44 70 67 70 64 69 68 70 66 55 16 20 20 mcaa⁴ R S E D TA V Y Y C A R A P G rel. oomcaa⁵ 89% 96% 63% 100% 96% 100% 91% 99% 97%100% 94% 79% 24% 29% 29% pos occupied⁶  4  2  2  1  4  1  5  2  2  1  3 8 10 14 18 CDR III amino acid¹ 98 99 100 A B C D E F G H I J K 101 A  1 1  4  1  2  2  1  1  1  1  1  2  1 B C  1 16  2  1  1  7  2  1 D  3  5 4  3  4  1  1 14 59 E  2  1  1  1 F  3  2  3  1  2  2  1 28  2 G 10 14 5 20 15 16  3  3  4 15  1  1  7 H  1  1  1  1 I  2  2  2  2  1  1  1 K 1  1 L  5  2  1  1  4  2  1  1  1 M  1  1  1  1 10 N  1  2  1  2  2  2 2  1  1  4 P  1  3  2  2  2  4  2  1  4  1  1  1 Q  1  1  1  1 R  8  1 4  2  1 16 S  5  5 21  5 11  8  4  3  2  1  2  1 T  1  3  4  2  5  2  1 1  1 V  3  3  3  4  2  2  2  1  2  1 W  3  1  1  2  3  1  5  1 X Y 20 5  4  9  1  2 11 20 10  6  9 10  7  1 Z —  2  3  6 11 11 14 23 26 26 3134 46 39 21  1 unknown (?)  1  1  1  2  3 not sequenced  4  4  4  4  5 5  5  5  5  5  5  5  5  5  5 sum of seq² 66 66 66 66 65 65 65 65 65 6565 65 65 65 65 oomcaa³ 20 16 21 20 15 16 23 26 26 31 34 46 39 28 59mcaa⁴ Y C S G — — — — — — — — — F D rel. oomcaa⁵ 30% 24% 32% 30% 23% 25%35% 40% 40% 48% 52% 71% 60% 43% 91% pos occupied⁶ 15 18 15 15 17 17 1512 11 11 10  8  7  6 6 Framework IV amino acid¹ 102 103 104 105 106 107108 109 110 111 112 113 sum A 670 B C 165 D  1  1 308 E  1  1 297 F  2226 G 58 59  1  1 928 H  1 14 I  3  4 286 K  3  1 325 L  3  1 40  1 386M  1  3 189 N  1 176 P  5  1 238 Q 52 494 R  1 351 S 53 51 972 T 54 11 1 51  1 736 V 15  1  1 54 54  1 699 W 59  1 243 X Y 34  1 542 Z  3 —  1578 unknown (?)  8 not sequenced  5  9  9 10 11 14 14 14 15 16 16 17 406sum of seq² 65 61 61 60 59 56 56 56 55 54 54 53 oomcaa³ 34 59 58 52 5954 40 54 51 54 53 51 mcaa⁴ Y W G Q G T L V T V S S rel. oomcaa⁵ 52% 97%95% 87% 100% 96% 71% 96% 93% 100% 98% 96% pos occupied⁶  9  3  4  7  1 3  5  3  2  1  2  3

TABLE 6B Analysis of V heavy chain subgroup 1B Frame work I amino acid¹1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 A 32 B C D E 1 5 1 35 F G 27 35 H 11 I K 3 1 34 33 L 3 26 1 M 1 1 N P 1 33 Q 21 20 26 R 1 1 2 S 27 T 1 1 V3 21 20 35 W X Y Z — unknown (?) not sequenced 15 15 15 13 13 13 13 13 65 5 5 5 5 5 sum of seq² 25 25 25 27 27 27 27 27 34 35 35 35 35 35 35oomcaa³ 21 21 20 26 20 26 27 27 32 35 35 34 33 33 35 mcaa⁴ Q V Q L V Q SG A E V K K P G rel. oomcaa⁵ 84% 84% 80% 96% 74% 96% 100% 100% 94% 100%100% 97% 94% 94% 100% pos occupied⁶ 3 3 4 2 4 2 1 1 3 1 1 2 2 3 1 Framework I amino acid¹ 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 A 34 30B C 35 D E 3 F 2 39 G 1 40 1 H I 1 1 1 K 33 28 L 1 M N 1 1 P 1 Q 2 R 2 2S 1 34 35 40 5 2 T 2 3 32 34 V 35 34 1 1 1 W X Y 36 Z — unknown (?) notsequenced 5 5 5 5 5 5 5 5 5 sum of seq² 35 35 35 35 35 35 35 35 35 40 4040 40 40 40 oomcaa³ 34 34 35 33 34 35 35 28 30 40 40 36 32 39 34 mcaa⁴ AS V K V S C K A S G Y T F T rel. oomcaa⁵ 97% 97% 100% 94% 97% 100% 100%80% 86% 100% 100% 90% 80% 98% 85% pos occupied⁶ 2 2 1 2 2 1 1 4 4 1 1 44 2 6 CDRI Frame work II amino acid¹ 31 A B 32 33 34 35 36 37 38 39 4041 42 43 A 2 6 39 B C D 1 5 1 1 E 1 1 F 2 2 G 14 1 1 39 H 3 1 34 I 9 K 1L 1 5 2 1 M 23 N 3 1 3 P 1 1 34 Q 1 1 1 1 39 39 R 1 37 1 S 15 2 1 1 T 14 V 1 2 2 38 W 40 X Y 1 32 19 1 Z — 40 40 unknown (?) not sequenced sumof seq² 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 oomcaa³ 15 40 40 3219 23 34 40 38 37 39 39 34 39 39 mcaa⁴ S — — Y Y M H W V R Q A P G Qrel. oomcaa⁵ 38% 100% 100% 80% 48% 58% 85% 100% 95% 93% 98% 98% 85% 98%98% pos occupied⁶ 10 1 1 5 11 5 5 1 2 4 2 2 4 2 2 Frame work II CDR IIamino acid¹ 44 45 46 47 48 49 50 51 52 A B C 53 54 55 A 1 1 7 1 B C D 11 E 39 1 1 F 2 1 1 G 28 39 1 1 9 1 39 H 2 I 3 34 K 1 L 37 1 M 37 2 4 N35 20 12 1 P 1 31 Q 1 R 10 4 3 1 S 1 2 1 20 T 1 3 V 1 1 W 40 33 X Y 2 Z— 40 40 unknown (?) not sequenced sum of seq² 40 40 40 40 40 40 40 40 4040 40 40 40 40 40 oomcaa³ 28 37 39 40 37 39 33 34 35 31 40 40 20 20 39mcaa⁴ G L E W M G W I N P — — N S G rel. oomcaa⁵ 70% 93% 98% 100% 93%98% 83% 85% 88% 78% 100% 100% 50% 50% 98% pos occupied⁶ 4 3 2 1 2 2 4 45 4 1 1 9 8 2 CDR II Framework III amino acid¹ 56 57 58 59 60 61 62 6364 65 66 67 68 69 70 A 1 2 27 2 1 1 B C D 1 4 E 2 2 1 1 F 4 39 3 G 15 61 34 H 1 1 I 1 1 1 1 13 K 2 2 8 36 1 L 1 1 1 M 23 N 17 18 1 P Q 36 37 R2 1 2 37 S 1 2 11 1 T 35 2 1 1 39 40 V 1 38 W 3 X Y 33 Z — unknown (?)not sequenced sum of seq² 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40oomcaa³ 17 35 18 33 27 36 36 39 37 34 37 38 39 23 40 mcaa⁴ N T N Y A Q KF Q G R V T M T rel. oomcaa⁵ 43% 88% 45% 83% 68% 90% 90% 98% 93% 85% 93%95% 98% 58% 100% pos occupied⁶ 8 4 8 4 4 4 5 2 3 4 2 3 2 4 1 FrameworkIII amino acid¹ 71 72 73 74 75 76 77 78 79 80 81 82 A B C A 2 12 35 B CD 35 1 4 E 1 35 F 1 G 1 1 2 H 1 I 22 1 K 1 L 2 39 39 M 1 1 37 1 N 4 7 12 P 3 Q R 34 1 4 2 16 S 1 37 27 1 35 20 T 1 38 5 1 39 1 V 4 1 1 W X Y 39Z — unknown (?) not sequenced sum of seq² 40 40 40 40 40 40 40 40 40 4040 40 40 40 40 oomcaa³ 34 35 38 37 22 27 39 35 39 37 35 39 35 20 39mcaa⁴ R D T S I S T A Y M E L S S L rel. oomcaa⁵ 85% 88% 95% 93% 55% 68%98% 88% 98% 93% 88% 98% 88% 50% 98% pos occupied⁶ 6 3 3 2 4 5 2 3 2 3 32 5 4 2 Framework III CDR III amino acid¹ 83 84 85 86 87 88 89 90 91 9293 94 95 96 97 A 1 2 40 37 1 6 1 B C 37 1 D 19 40 1 7 5 E 19 2 1 F 2 2 11 1 G 1 7 7 5 H 1 I 1 1 1 1 K 1 1 1 L 2 1 2 4 4 M 2 2 N 1 P 1 1 6 4 Q 1R 37 1 31 5 1 S 1 36 1 1 1 3 3 1 T 1 40 2 1 1 2 V 33 1 7 1 1 W 1 1 X Y38 35 5 5 Z — 1 1 unknown (?) not sequenced 1 1 1 1 1 1 3 3 3 sum ofseq² 40 40 40 40 40 40 39 39 39 39 39 39 37 37 37 oomcaa³ 37 36 19 40 4040 33 38 35 37 37 31 7 7 5 mcaa⁴ R S D D T A V Y Y C A R D G D rel.oomcaa⁵ 93% 90% 48% 100% 100% 100% 85% 97% 90% 95% 95% 79% 19% 19% 14%pos occupied⁶ 4 4 3 1 1 1 5 2 4 3 3 8 10 12 18 CDR III amino acid¹ 98 99100 A B C D E F G H I J K 101 A 1 2 3 1 3 1 5 B C 3 2 1 D 2 3 1 5 4 1 22 1 2 27 E 1 1 2 1 1 F 3 2 1 1 1 1 2 15 G 5 9 4 7 1 3 2 2 1 1 3 1 H 2 11 I 3 1 1 1 1 1 1 1 K 1 1 1 1 1 L 4 3 1 2 1 1 2 1 2 M 1 1 1 4 N 1 1 1 13 1 1 P 1 1 3 2 1 Q 1 2 1 R 1 3 1 1 1 S 4 3 6 3 2 2 1 1 T 2 1 5 1 1 1 11 1 V 1 3 1 2 1 1 2 1 1 W 2 2 1 1 1 4 X Y 4 2 3 4 3 3 2 1 2 5 6 2 Z — 46 8 10 11 14 20 23 25 25 25 23 18 11 6 unknown (?) 3 not sequenced 3 3 34 4 4 4 4 4 4 4 4 4 4 4 sum of seq² 37 37 37 36 36 36 36 36 36 36 36 3636 36 36 oomcaa³ 5 9 8 10 11 14 20 23 25 25 25 23 18 15 27 mcaa⁴ G G — —— — — — — — — — — F D rel. oomcaa⁵ 14% 24% 22% 28% 31% 39% 56% 64% 69%69% 69% 64% 50% 42% 75% pos occupied⁶ 13 13 12 12 17 14 13 10 9 8 7 8 85 5 Framework IV amino acid¹ 102 103 104 105 106 107 108 109 110 111 112113 sum A 340 B C 79 D 2 179 E 1 159 F 1 130 G 27 26 1 450 H 1 51 I 7 3113 K 2 194 L 12 1 204 M 2 144 N 1 138 P 1 1 128 Q 23 253 R 1 247 S 3 118 18 432 T 21 6 16 1 390 V 6 21 18 342 W 29 158 X Y 11 294 Z — 3 394unknown (?) 3 not sequenced 4 11 13 13 14 19 19 19 20 20 21 22 458 sumof seq² 36 29 27 27 26 21 21 21 20 20 19 18 oomcaa³ 11 29 27 23 26 21 1221 16 18 18 18 mcaa⁴ Y W G Q G T L V T V S S rel. oomcaa⁵ 31% 100% 100%85% 100% 100% 57% 100% 80% 90% 95% 100% pos occupied⁶ 10 1 1 4 1 1 4 1 33 2 1

TABLE 6C Analysis of V heavy chain subgroup 2 Frame work I amino acid¹ 12 3 4 5 6 7 8 9 10 11 12 13 14 15 A 3 B C D E 1 6 F G 6 H I 1 K 3 6 1 L6 6 M N 1 P 1 6 6 Q 2 R 2 S 4 T 6 1 2 5 V 5 1 6 W X Y Z 3 — unknown (?)not sequenced 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 sum of seq² 6 6 6 6 6 6 6 66 6 6 6 6 6 6 oomcaa³ 3 5 6 6 3 6 4 6 6 3 6 6 6 6 5 mcaa⁴ Z V T L K E SG P A L V K P T rel. oomcaa⁵ 50% 83% 100% 100% 50% 100% 67% 100% 100%50% 100% 100% 100% 100% 83% pos occupied⁶ 3 2 1 1 3 1 3 1 1 3 1 1 1 1 2Frame work I amino acid¹ 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 A1 B C 7 D E 2 F 3 6 1 G 7 H I K L 6 6 2 1 6 M N P 1 Q 4 R S 1 6 6 6 T 56 6 6 1 V 2 W X Y 1 Z — unknown (?) not sequenced 1 1 1 1 1 1 sum ofseq² 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 oomcaa³ 4 5 6 6 6 6 7 6 3 6 7 6 6 6 6mcaa⁴ Q T L T L T C T F S G F S L S rel. oomcaa⁵ 67% 83% 100% 100% 100%100% 100% 86% 43% 86% 100% 86% 86% 86% 86% pos occupied⁶ 2 2 1 1 1 1 1 23 2 1 2 2 2 2 CDRI Frame work II amino acid¹ 31 A B 32 33 34 35 36 37 3839 40 41 42 43 A 1 1 B C 2 D 1 E F G 4 3 3 1 7 H I 1 7 K 6 L M 5 N 2 P 57 Q 6 R 2 1 7 1 1 S 2 4 4 1 T 3 1 V 2 7 W 7 X Y Z — unknown (?) notsequenced sum of seq² 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 oomcaa³ 3 4 4 5 3 74 7 7 7 6 5 7 7 6 mcaa⁴ T S G M G V S W I R Q P P G K rel. oomcaa⁵ 43%57% 57% 71% 43% 100% 57% 100% 100% 100% 86% 71% 100% 100% 86% posoccupied⁶ 3 4 3 2 4 1 2 1 1 1 2 3 1 1 2 Frame work II CDR II amino acid¹44 45 46 47 48 49 50 51 52 A B C 53 54 55 A 6 7 B C D 2 3 6 E 7 F 2 G 1H 2 1 I 6 K L 7 7 2 1 1 M N 3 P Q R 2 S 2 T V W 7 1 4 X 1 1 1 Y 1 1 Z —6 7 7 unknown (?) not sequenced sum of seq² 7 7 7 7 7 7 7 7 7 7 7 7 7 77 oomcaa³ 6 7 7 7 7 7 2 6 2 6 7 7 4 3 6 mcaa⁴ A L E W L A H I D — — — WD D rel. oomcaa⁵ 86% 100% 100% 100% 100% 100% 29% 86% 29% 86% 100% 100%57% 43% 86% pos occupied⁶ 2 1 1 1 1 1 4 2 5 2 1 1 3 3 2 CDR II FrameworkIII amino acid¹ 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 A B C D 5 E1 1 F 1 1 G H 1 I 6 K 1 6 4 L 7 7 M N P 2 Q R 2 1 2 7 S 2 6 7 4 1 5 T 43 6 2 V 1 W 1 X 1 Y 3 4 Z — unknown (?) not sequenced sum of seq² 7 7 77 7 7 7 7 7 7 7 7 7 7 7 oomcaa³ 5 6 3 4 6 4 7 7 4 4 7 7 6 6 5 mcaa⁴ D KY Y S T S L K S R L T I S rel.oomcaa⁵ 71% 86% 43% 57% 86% 57% 100% 100%57% 57% 100% 100% 86% 86% 71% pos occupied⁶ 3 2 3 4 2 3 1 1 3 2 1 1 2 22 Framework III amino acid¹ 71 72 73 74 75 76 77 78 79 80 81 82 A B C AB C D 6 1 E F 1 G H I 2 1 K 6 6 L 6 M 7 5 N 1 5 6 P Q 7 R 1 1 S 7 2 T 65 5 V 7 7 1 W X Y Z — 1 1 1 unknown (?) not sequenced sum of seq² 7 7 77 7 7 7 7 7 7 7 7 7 7 7 oomcaa³ 6 6 6 7 6 5 7 7 7 6 5 7 5 6 5 mcaa⁴ K DT S K N Q V V L T M T N M rel.oomcaa⁵ 86% 86% 86% 100% 86% 71% 100% 100%100% 86% 71% 100% 71% 86% 71% pos occupied⁶ 2 2 2 1 2 2 1 1 1 2 2 1 3 23 Framework III CDR III amino acid¹ 83 84 85 86 87 88 89 90 91 92 93 9495 96 97 A 1 5 5 B C 7 D 6 7 E F G 2 H 1 1 I 3 K L M N 1 1 2 P 7 1 1 Q 1R 6 1 S 1 T 7 7 1 V 6 2 1 1 1 W X Y 7 7 2 Z — unknown (?) not sequenced1 1 1 sum of seq² 7 7 7 7 7 7 7 7 7 7 7 7 6 6 6 oomcaa³ 6 7 6 7 7 5 7 77 7 5 6 3 1 2 mcaa⁴ D P V D T A T Y Y C A R I H N rel.oomcaa⁵ 86% 100%86% 100% 100% 71% 100% 100% 100% 100% 71% 86% 50% 17% 33% pos occupied⁶2 1 2 1 1 2 1 1 1 1 2 2 4 6 4 CDR III amino acid¹ 98 99 100 A B C D E FG H I J K 101 A 1 2 1 B C D 6 E 2 1 F 3 G 1 1 1 2 1 1 1 1 H I 2 K 1 L 11 1 M 1 2 N 1 P 1 1 Q R 1 S 1 1 T 1 1 V 1 1 1 W 1 1 1 X Y 1 2 1 1 1 2 Z— 2 2 3 4 4 4 6 5 3 unknown (?) not sequenced 1 1 1 1 1 1 1 1 1 1 1 1 11 1 sum of seq² 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 oomcaa³ 2 1 2 2 2 2 3 4 44 6 5 3 3 6 mcaa⁴ I G E A — — — — — — — — — F D rel. oomcaa⁵ 33% 17% 33%33% 33% 33% 50% 67% 67% 67% 100% 83% 50% 50% 100% pos occupied⁶ 5 6 5 54 5 3 3 3 3 1 2 3 3 1 Framework IV amino acid¹ 102 103 104 105 106 107108 109 110 111 112 113 sum A 1 35 B C 16 D 43 E 21 F 18 G 6 6 55 H 6 I29 K 1 1 42 L 1 3 78 M 20 N 23 P 1 1 41 Q 3 23 R 2 41 S 6 3 82 T 6 1 5102 V 3 6 6 68 W 6 29 X 4 Y 1 35 Z 3 — 56 unknown (?) not sequenced 1 11 1 1 1 1 1 1 1 1 4 54 sum of seq² 6 6 6 6 6 6 6 6 6 6 6 3 oomcaa³ 3 6 63 6 6 3 6 5 6 6 3 mcaa⁴ V W G Q G T L V T V S S rel. oomcaa⁵ 50% 100%100% 50% 100% 100% 50% 100% 83% 100% 100% 100% pos occupied⁶ 4 1 1 3 1 14 1 2 1 1 1

TABLE 6D Analysis of V heavy chain subgroup 3 Frame work I amino acid¹ 12 3 4 5 6 7 8 9 10 11 12 13 14 15 A 1 1 12 1 3 1 B 1 1 1 C D 1 1 16 E110 9 15 166 9 8 2 F 4 G 181 193 174 1 202 H 5 4 I 9 K 5 3 26 L 1 5 17643 140 1 M 12 1 N 1 P 1 194 Q 41 138 1 3 12 162 R 6 4 S 178 2 8 T 1 V 5147 1 118 62 195 W 1 X Y Z 8 — unknown (?) not sequenced 47 47 45 33 3232 32 31 10 7 6 6 6 6 6 sum of seq² 165 165 167 179 180 180 180 181 202205 206 206 206 206 206 oomcaa³ 110 147 138 176 118 166 178 181 193 174140 195 162 194 202 mcaa⁴ E V Q L V E S G G G L V Q P G rel. oomcaa⁵ 67%89% 83% 98% 66% 92% 99% 100% 96% 85% 68% 95% 79% 94% 98% pos occupied⁶ 54 7 4 5 4 3 1 2 5 3 4 7 4 4 Frame work I amino acid¹ 16 17 18 19 20 2122 23 24 25 26 27 28 29 30 A 183 192 1 B C 1 209 D 7 E 8 8 3 1 F 1 1 1201 201 G 134 2 207 3 H 1 I 2 3 17 1 K 15 4 L 205 201 6 3 M 1 1 N 10 10P 1 2 Q 1 R 62 191 11 S 206 207 4 2 209 15 174 T 4 1 2 4 4 1 163 V 8 7 91 6 W X Y Z — unknown (?) not sequenced 4 4 4 4 3 3 3 3 3 3 1 1 2 1 2sum of seq² 208 208 208 208 209 209 209 209 209 209 211 211 210 211 210oomcaa³ 134 206 205 191 201 207 209 183 192 209 207 201 163 201 174mcaa⁴ G S L R L S C A A S G F T F S rel. oomcaa⁵ 64% 99% 99% 92% 96% 99%100% 88% 92% 100% 98% 95% 78% 95% 83% pos occupied⁶ 4 3 4 3 2 3 1 7 5 13 4 8 4 7 CDRI Frame work II amino acid¹ 31 A B 32 33 34 35 36 37 38 3940 41 42 43 A 1 17 80 1 1 187 1 B C 1 1 D 26 3 7 2 E 1 10 1 1 F 5 G 1331 1 2 209 H 4 88 I 1 1 15 12 K 7 1 202 L 3 3 2 3 1 2 1 M 193 N 35 8 334 P 1 1 4 191 Q 209 1 1 R 7 207 7 8 S 103 17 8 72 3 14 T 9 15 10 4 5 V2 7 1 197 2 W 30 212 X 1 Y 1 154 19 3 Z — 210 210 unknown (?) notsequenced 2 2 2 1 1 1 sum of seq² 210 210 210 210 210 212 212 212 211211 211 212 212 212 212 oomcaa³ 103 210 210 154 80 193 88 212 197 207209 187 191 209 202 mcaa⁴ S — — Y A M H W V R Q A P G K rel. oomcaa⁵ 49%100% 100% 73% 38% 91% 42% 100% 93% 98% 99% 88% 90% 99% 95% pos occupied⁶14 1 1 9 10 4 9 1 3 3 3 9 5 4 4 Frame work II CDR II amino acid¹ 44 4546 47 48 49 50 51 52 A B C 53 54 55 A 1 77 42 1 2 14 7 B 3 1 C 1 D 1 794 8 3 E 198 3 2 1 2 1 F 7 1 2 1 1 8 G 207 33 11 10 46 4 163 85 H 6 1 I3 3 191 1 1 K 1 37 2 30 3 L 211 5 12 1 M 1 1 N 13 7 9 2 13 11 1 P 1 1 1Q 7 7 10 R 1 24 1 17 5 1 2 16 S 3 1 102 11 9 118 43 1 74 17 82 T 3 5 4 213 12 3 3 V 3 204 49 2 1 6 W 210 1 8 6 X 4 3 Y 1 22 5 58 8 Z — 14 178178 2 1 1 unknown (?) not sequenced sum of seq² 212 212 212 212 212 212212 212 212 212 212 212 212 212 212 oomcaa³ 207 211 198 210 204 102 49191 118 58 178 178 94 163 85 mcaa⁴ G L E W V S V I S Y — — D G G rel.oomcaa⁵ 98% 100% 93% 99% 96% 48% 23% 90% 56% 27% 84% 84% 44% 77% 40% posoccupied⁶ 4 2 5 3 3 3 15 9 11 19 5 5 12 9 12 CDR II Framework III aminoacid¹ 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 A 9 1 2 174 33 1 B 12 C D 11 17 160 E 8 3 2 1 2 F 1 3 2 207 G 5 1 5 4 5 212 1 H 1 4 I 3 37 28 14 208 K 1 61 199 8 L 1 1 1 1 1 1 M 8 2 1 N 51 4 2 2 P 1 1 6 8 18 1 Q3 2 2 2 R 5 4 5 6 201 S 48 11 4 193 2 7 211 T 42 97 5 7 189 1 V 2 10 2204 1 3 W 2 X 4 1 1 Y 9 151 210 1 1 1 Z — unknown (?) not sequenced sumof seq² 212 212 212 212 212 212 212 212 212 212 212 212 212 212 212oomcaa³ 51 97 151 210 174 160 193 204 199 212 201 207 189 208 211 mcaa⁴N T Y Y A D S V K G R F T I S rel. oomcaa⁵ 24% 46% 71% 99% 82% 75% 91%96% 94% 100% 95% 98% 89% 98% 100% pos occupied⁶ 19 12 15 2 9 8 3 2 6 1 45 5 3 2 Framework III amino acid¹ 71 72 73 74 75 76 77 78 79 80 81 82 AB C A 57 1 8 1 B 2 C D 199 38 2 2 1 10 E 6 4 5 F 13 G 1 4 H 1 1 2 2 I 12 2 3 1 1 K 186 6 3 L 188 209 3 1 212 M 1 2 10 3 2 205 N 5 170 2 188 3181 10 P 1 Q 7 199 R 211 1 1 2 8 S 153 8 10 56 3 6 186 T 142 1 4 2 V 111 1 1 W X 2 2 4 1 Y 194 Z — unknown (?) not sequenced 1 1 sum of seq²212 212 211 211 212 212 212 212 212 212 212 212 212 212 212 oomcaa³ 211199 170 153 186 188 142 188 194 209 199 205 181 186 212 mcaa⁴ R D N S KN T L Y L Q M N S L rel. oomcaa⁵ 100% 94% 81% 73% 88% 89% 67% 89% 92%99% 94% 97% 85% 88% 100% pos occupied⁶ 2 4 4 3 8 7 6 5 5 3 6 4 11 7 1Framework III CDR III amino acid¹ 83 84 85 86 87 88 89 90 91 92 93 94 9596 97 A 149 1 1 207 173 2 15 9 11 B C 1 210 5 2 1 D 5 15 209 2 54 7 6 E1 190 11 2 11 F 1 15 1 9 6 G 1 1 6 4 1 2 8 34 26 35 H 1 1 3 11 I 8 2 415 10 K 30 60 4 3 5 L 18 1 6 11 7 M 2 1 6 1 N 1 1 2 20 4 3 P 9 1 3 4 2910 Q 1 5 3 9 2 R 177 103 9 30 19 S 1 1 3 9 8 11 T 3 28 207 1 25 15 7 620 V 9 187 10 1 7 7 15 W 1 3 4 3 X 1 Y 211 194 12 9 8 Z — 1 3 4 unknown(?) not sequenced 1 1 1 1 1 1 1 1 7 12 13 sum of seq² 212 212 212 212211 211 211 211 211 211 211 211 205 200 199 oomcaa³ 177 149 190 209 207207 187 211 194 210 173 103 54 30 35 mcaa⁴ R A E D T A V Y Y C A R D R Grel. oomcaa⁵ 83% 70% 90% 99% 98% 98% 89% 100% 92% 100% 82% 49% 26% 15%18% pos occupied⁶ 5 10 4 4 4 2 7 1 4 2 5 14 18 20 21 CDR III amino acid¹98 99 100 A B C D E F G H I J K 101 A 7 13 7 9 6 2 3 5 5 9 13 2 B C 13 51 2 11 3 2 1 D 11 7 10 4 2 3 10 3 3 1 3 2 146 E 6 3 1 13 1 1 1 F 3 5 4 55 6 3 5 7 2 1 1 65 1 G 34 17 35 17 14 23 10 5 1 5 3 2 32 6 H 3 4 3 2 9 21 3 1 2 8 1 I 6 11 4 4 3 1 3 10 3 3 2 1 2 K 2 11 3 1 L 26 13 4 12 8 2 63 10 3 2 1 M 1 2 1 32 N 4 6 4 3 2 2 6 2 5 2 P 6 5 5 6 9 8 2 3 2 1 3 9 Q4 1 1 1 1 1 1 R 4 10 9 7 5 5 2 3 1 1 2 4 S 16 28 27 25 24 8 11 9 3 2 3 11 1 T 6 12 9 17 17 1 2 5 1 9 3 1 V 13 7 15 4 3 6 2 12 1 1 1 1 W 6 5 6 72 4 1 6 10 X 1 1 Y 16 14 17 5 8 18 20 13 20 25 28 32 28 Z — 12 21 35 5473 87 102 110 126 135 134 120 91 71 21 unknown (?) 3 2 1 1 3 2 notsequenced 14 14 14 14 15 19 21 22 23 23 23 25 25 26 25 sum of seq² 198198 198 197 196 192 190 189 188 188 188 186 186 185 186 oomcaa³ 34 28 3554 73 87 102 110 126 135 134 120 91 71 146 mcaa⁴ G S G — — — — — — — — —— — D rel. oomcaa⁵ 17% 14% 18% 27% 37% 45% 54% 58% 67% 72% 71% 65% 49%38% 78% pos occupied⁶ 20 20 19 20 19 20 17 14 14 12 12 13 12 8 11Framework IV amino acid¹ 102 103 104 105 106 107 108 109 110 111 112 113sum A 1 1 2 1767 B 1 13 C 470 D 2 1121 E 1 832 F 2 807 G 140 130 1 2743H 4 179 I 15 1 1 651 K 13 933 L 10 1 91 2 1881 M 6 496 N 1 1 844 P 17 11 568 Q 111 949 R 8 1413 S 7 1 118 110 3009 T 123 27 122 1 1426 V 34 1 1125 119 1851 W 158 686 X 26 Y 82 1598 Z 8 — 9 2 2 2 2 2 2 2 2 2 1 1 2023unknown (?) 12 not sequenced 27 50 67 75 78 81 83 84 86 89 92 97 1650sum of seq² 184 161 144 136 133 130 128 127 125 122 119 114 oomcaa³ 82158 140 111 130 123 91 125 122 119 118 110 mcaa⁴ Y W G Q G T L V T V S Srel. oomcaa⁵ 45% 98% 97% 82% 98% 95% 71% 98% 98% 98% 99% 96% posoccupied⁶ 12 3 4 6 3 6 6 2 3 3 2 4

TABLE 6E Analysis of V heavy chain subgroup 4 Frame work I amino acid¹ 12 3 4 5 6 7 8 9 10 11 12 13 14 15 A 19 1 B C D E 32 F G 54 1 53 H 4 2 IK 1 54 L 7 54 53 19 1 M N P 33 51 1 Q 52 50 51 20 R 1 S 33 52 T 1 V 47 134 W 20 X Y Z 1 — unknown (?) not sequenced 3 3 3 3 4 4 4 3 3 4 4 3 3 44 sum of seq² 54 54 54 54 53 53 53 54 54 53 53 54 54 53 53 oomcaa³ 52 4750 54 51 32 33 54 33 53 53 34 54 51 52 mcaa⁴ Q V Q L Q E S G P G L V K PS rel. oomcaa⁵ 96% 87% 93% 100% 96% 60% 62% 100% 61% 100% 100% 63% 100%96% 98% pos occupied⁶ 3 2 2 1 2 3 2 1 4 1 1 3 1 3 2 Frame work I aminoacid¹ 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 A 1 1 22 B C 53 D 1 E44 F 1 22 G 2 53 53 H 1 I 1 1 32 K 1 L 53 50 M N 1 P 2 3 Q 7 R 1 3 S 522 35 51 1 52 T 52 53 29 V 1 55 1 1 W X Y 19 1 Z — unknown (?) notsequenced 4 4 4 3 4 4 4 2 2 2 2 2 2 1 1 sum of seq² 53 53 53 54 53 53 5355 55 55 55 55 55 56 56 oomcaa³ 44 52 53 52 50 53 53 29 55 35 53 53 5132 52 mcaa⁴ E T L S L T C T V S G G S I S rel. oomcaa⁵ 83% 98% 100% 96%94% 100% 100% 53% 100% 64% 96% 96% 93% 57% 93% pos occupied⁶ 3 2 1 3 3 11 5 1 3 3 3 3 4 3 CDRI Frame work II amino acid¹ 31 A B 32 33 34 35 3637 38 39 40 41 42 43 A 1 8 1 B C 1 D 4 1 1 1 1 E 1 F 1 1 1 G 21 3 4 8 55H 2 2 I 51 K 54 L 1 1 M N 1 2 2 1 P 50 49 Q 1 56 R 2 1 57 3 S 25 5 9 144 1 3 T 2 1 3 1 1 V 3 W 1 2 56 57 X Y 48 52 Z — 45 39 unknown (?) notsequenced 1 1 1 1 sum of seq² 56 56 56 56 56 56 57 57 57 57 57 57 57 5757 oomcaa³ 25 45 39 48 52 56 44 57 51 57 56 50 49 55 54 mcaa⁴ S — — Y YW S W I R Q P P G K rel. oomcaa⁵ 45% 80% 70% 86% 93% 100% 77% 100% 89%100% 98% 88% 86% 96% 95% pos occupied⁶ 7 6 6 7 4 1 5 1 5 1 2 5 2 3 2Frame work II CDR II amino acid¹ 44 45 46 47 48 49 50 51 52 A B C 53 5455 A 1 B C D 1 1 E 56 22 F 1 1 G 55 56 1 1 57 H 24 I 54 1 54 K L 55 2 MN 21 P 2 Q 1 1 R 2 9 1 S 7 1 52 T 8 5 V 1 3 W 56 X Y 1 15 32 23 Z — 5757 57 unknown (?) not sequenced sum of seq² 57 57 57 57 57 57 57 57 5757 57 57 57 57 57 oomcaa³ 55 55 56 56 54 56 22 54 32 57 57 57 24 52 57mcaa⁴ G L E W I G E I Y — — — H S G rel. oomcaa⁵ 96% 96% 98% 98% 95% 98%39% 95% 56% 100% 100% 100% 42% 91% 100% pos occupied⁶ 2 2 2 2 3 2 8 2 61 1 1 5 2 1 CDR II Framework III amino acid¹ 56 57 58 59 60 61 62 63 6465 66 67 68 69 70 A 1 1 1 B C D 2 1 E F 3 G 1 1 H 2 I 1 1 1 1 48 K 1 53L 1 55 1 M 7 N 2 40 53 2 P 54 1 Q R 2 3 56 S 49 1 2 56 56 1 56 T 1 54 11 1 51 1 V 1 1 53 2 W X Y 11 54 Z — unknown (?) not sequenced 1 1 1 1 11 sum of seq² 57 57 57 57 56 56 56 56 57 57 57 56 56 57 57 oomcaa³ 49 5440 54 53 54 56 55 53 56 56 53 51 48 56 mcaa⁴ S T N Y N P S L K S R V T IS rel. oomcaa⁵ 86% 95% 70% 95% 95% 96% 100% 98% 93% 98% 98% 95% 91% 84%98% pos occupied⁶ 7 4 6 2 3 3 1 2 3 2 2 4 5 3 2 Framework III aminoacid¹ 71 72 73 74 75 76 77 78 79 80 81 82 A B C A 1 1 B C D 55 1 E 1 1 F1 54 1 G 1 H I 3 1 1 K 1 51 3 46 2 L 3 1 3 1 55 53 2 M 2 1 1 1 N 1 54 33 1 P Q 1 54 1 1 R 2 2 2 S 1 57 1 57 2 1 44 55 T 52 1 4 V 50 1 2 54 W XY Z — unknown (?) not sequenced sum of seq² 57 57 57 57 57 57 57 57 5757 57 57 57 57 57 oomcaa³ 50 55 52 57 51 54 54 54 57 55 46 53 44 55 54mcaa⁴ V D T S K N Q F S L K L S S V rel. oomcaa⁵ 88% 96% 91% 100% 89%95% 95% 95% 100% 96% 81% 93% 77% 96% 95% pos occupied⁶ 4 3 5 1 6 2 2 4 13 8 4 7 3 3 Framework III CDR III amino acid¹ 83 84 85 86 87 88 89 90 9192 93 94 95 96 97 A 55 57 57 56 3 3 3 B C 57 1 D 57 6 5 E 6 1 1 F 4 1 G25 9 10 H 1 I 3 1 K 2 1 L 1 2 6 7 M 1 1 4 N 3 P 4 5 Q 1 R 1 54 4 12 2 S1 2 1 1 1 4 8 T 53 55 1 1 2 1 V 1 55 1 1 4 2 2 W 1 2 1 X Y 57 56 1 4 Z —unknown (?) not sequenced 1 1 1 sum of seq² 57 57 57 57 57 57 57 57 5757 57 57 56 56 56 oomcaa³ 53 55 57 57 55 57 55 57 56 57 56 54 25 12 10mcaa⁴ T A A D T A V Y Y C A R G R G rel. oomcaa⁵ 93% 96% 100% 100% 96%100% 96% 100% 98% 100% 98% 96% 45% 21% 18% pos occupied⁶ 3 3 1 1 2 1 3 12 1 2 4 12 16 16 CDR III amino acid¹ 98 99 100 A B C D E F G H I J K 101A 2 5 4 2 2 4 2 1 1 1 12 B C 1 D 5 5 4 3 2 4 3 1 1 2 1 41 E 2 1 1 3 1 21 F 1 2 3 2 2 1 1 31 G 8 10 11 4 7 7 6 1 1 1 2 1 9 H 1 1 1 2 I 2 4 1 3 23 1 1 K 2 2 1 L 3 5 3 2 4 1 5 3 3 1 M 3 1 2 1 9 N 2 1 1 5 1 1 2 P 3 1 12 1 1 1 2 3 1 2 1 Q 1 1 1 1 3 1 R 5 5 3 2 3 1 2 2 1 S 8 1 2 5 7 4 2 1 11 T 3 4 4 3 3 1 1 1 V 5 4 4 7 3 1 2 1 W 2 2 4 5 1 1 2 2 1 3 2 X Y 5 3 64 2 3 4 8 4 8 3 5 8 2 Z — 1 2 4 6 9 11 16 23 27 29 34 31 14 4 unknown(?) 1 1 1 1 not sequenced 1 1 2 3 3 6 7 8 9 9 10 11 11 11 11 sum of seq²56 56 55 54 54 51 50 49 48 48 47 46 46 46 46 oomcaa³ 8 10 11 7 9 11 1623 27 29 34 31 14 31 41 mcaa⁴ G G G V — — — — — — — — — F D rel. oomcaa⁵14% 18% 20% 13% 17% 22% 32% 47% 56% 60% 72% 67% 30% 67% 89% posoccupied⁶ 16 16 16 16 18 18 13 15 13 10 9 8 5 4 4 Framework IV aminoacid¹ 102 103 104 105 106 107 108 109 110 111 112 113 sum A 1 1 332 B C113 D 210 E 176 F 135 G 41 40 1 674 H 1 1 45 I 9 1 282 K 3 278 L 4 19540 M 9 43 N 1 204 P 3 2 2 281 Q 29 334 R 1 4 1 250 S 1 1 36 33 986 T 133 8 34 532 V 12 36 36 488 W 46 267 X Y 16 455 Z 1 — 466 unknown (?) 4not sequenced 10 11 16 17 17 20 20 21 21 21 21 22 426 sum of seq² 47 4641 40 40 37 37 36 36 36 36 35 oomcaa³ 16 46 41 29 40 33 19 36 34 36 3633 mcaa⁴ Y W G Q G T L V T V S S rel. oomcaa⁵ 34% 100% 100% 73% 100% 89%51% 100% 94% 100% 100% 94% pos occupied⁶ 8 1 1 6 1 5 4 1 3 1 1 2

TABLE 6F Analysis of V heavy chain subgroup 5 Framework I amino acid¹ 12 3 4 5 6 7 8 9 10 11 12 13 14 15 A  1  1 89  1  1 B C  1 D  2 E 88  1 2  4 93 F G  1 92 94 H I K 94 94 L  1 91  2 M  3 N P  1  1 94 Q  3 92 1 90 R  1  1  1  1 S 92 T V 90 89  1 91 W X Y Z — unknown (?) notsequenced  5  5  5  5  4  4  4  4  2  2  2  2  2  2  2 sum of seq² 92 9292 92 93 93 93 93 95 95 95 95 95 95 95 oomcaa³ 88 90 92 91 89 90 92 9289 93 91 94 94 94 94 mcaa⁴ E V Q L V Q S G A E V K K P G rel. oomcaa⁵96% 98% 100% 99% 96% 97% 99% 99% 94% 98% 96% 99% 99% 99% 99% posoccupied⁶  3  3  1  2  4  3  2  2  4  2  3  2  2  2  2 Framework I aminoacid¹ 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 A  3  2  4 B C 96  1D  2 E 92  2 F  1  3  6 97 G 92 93 H I 96  4 K 77 89  1 L 95 M  1  1 N 1  2  4 P  1 Q  3  1  4 R 17  1  1  2 S 94 94  1 90 84 10 T  2  5 75 VW X Y 90 Z — unknown (?) not sequenced  2  2  2  1  1  1  1  1  1  1  1 1 sum of seq² 95 95 95 96 96 96 96 96 96 96 96 96 97 97 97 oomcaa³ 9294 95 77 96 94 96 89 92 90 93 90 84 97 75 mcaa⁴ E S L K I S C K G S G YS F T rel. oomcaa⁵ 97% 99% 100% 80% 100% 98% 100% 93% 96% 94% 97% 94%87% 100% 77% pos occupied⁶  2  2  1  4  1  2  1  5  3  4  3  2  7  1  5CDRI Framework II amino acid¹ 31 A B 32 33 34 35 36 37 38 39 40 41 42 43A  8  1  1 B C  1 D  2  1 E  1  3 F  2 G  1 72 97 H  1  4  1 I 93 K  194 L  1  2 M  1  1 92 N 14  2 P  1 96 Q 97 R  1 95  1 S 61  2  2 15 T 16 2  1  1 V  1 93  2 W 93 97 X Y 87 Z — 97 97 unknown (?) not sequencedsum of seq² 97 97 97 97 97 97 97 97 97 97 97 97 97 97 97 oomcaa³ 61 9797 87 93 93 72 97 93 95 97 92 96 97 94 mcaa⁴ S — — Y W I G W V R Q M P GK rel. oomcaa⁵ 63% 100% 100% 90% 96% 96% 74% 100% 96% 98% 100% 95% 99%100% 97% pos occupied ⁶  8  1  1  5  4  4  5  1  4  3  1  5  2  1  2Framework II CDR II amino acid¹ 44 45 46 47 48 49 50 51 52 A B C 53 5455 A  1  1  2  1 B C  1  1 D 14  8 93 E 97  2 F  1  2 G 96 95 69  1 H  3 1 I  1 75 92 K L 94  2  2  1 M 89  1 N P  2  1 93  1 Q  1 R  1 14  1 S 1  1 16 96 T  3  1  1 V  5  1  1  2 W 94 X Y  3 76 Z — unknown (?) notsequenced sum of seq² 97 97 97 97 97 97 97 97 97 97 97 97 97 97 97oomcaa³ 96 94 97 94 89 95 75 92 76 93 97 97 69 93 96 mcaa⁴ G L E W M G II Y P — — G D S rel. oomcaa⁵ 99% 97% 100% 97% 92% 98% 77% 95% 78% 96%100% 100% 71% 96% 99% pos occupied⁶  2  3  1  2  4  3  7  5  6  5  1  1 6  4  2 CDR II Framework III amino acid¹ 56 57 58 59 60 61 62 63 64 6566 67 68 69 70 A  6  1 B C  1  1 D 77  2 E  3  2 F  2 91  1  3 G  1 94 H15 I  4  1  1  3 88 K  2 L  1  4  2 M  3 N  2 14  2 P 95  1  1 Q 91 81 R78  3  1  1 S  2  2 95  1 95  1  1 95 T 85  2  1 96 V  1 93  2 W X Y 1292 Z — unknown (?) not sequenced sum of seq² 97 97 97 97 97 97 97 97 9797 97 97 97 97 97 oomcaa³ 77 85 78 92 95 95 95 91 91 94 81 93 96 88 95mcaa⁴ D T R Y S P S F Q G Q V T I S rel. oomcaa⁵ 79% 88% 80% 95% 98% 98%98% 94% 94% 97% 84% 96% 99% 91% 98% pos occupied⁶  6  4  5  4  3  3  3 4  4  3  3  3  2  5  2 Framework III amino acid¹ 71 72 73 74 75 76 7778 79 80 81 82 A B C A 88  1 91 B C  1 D 97  1 E  2  1 F  1 G  3  1 H  3I 91 K 93 L 96 97 M  1 N  7  2  2 P  1  1 Q  1 93 R  1  1  1  1  3 S 96 1 87  2  1  1 90 91 T  4  2 94  2  1 V  9  2  1 W 95 X Y 94 Z — unknown(?) not sequenced sum of seq² 97 97 97 97 97 97 97 97 97 97 97 97 97 9797 oomcaa³ 88 97 93 96 91 87 94 91 94 96 93 95 90 91 97 mcaa⁴ A D K S IS T A Y L Q W S S L rel. oomcaa⁵ 91% 100% 96% 99% 94% 90% 97% 94% 97%99% 96% 98% 93% 94% 100% pos occupied⁶  2  1  4  2  4  4  3  5  4  2  3 3  5  4  1 Framework III CDR III amino acid¹ 83 84 85 86 87 88 89 90 9192 93 94 95 96 97 A  1 96 93 92  1  1  2 B C 95 D 96  3  3 E  1  1  1  1F  2  6  1 G  4  1  9 11 H 10  1 I  2  9  3 K 91  1  1  1  1 L  2 11  2 3 M 84  2 N  2  1 P  5  1  4 Q  1  3  2 R  3 92  7  9  2 S 96  5  1  1 3  2 T  1  1  1 88  1  1  1  3  2 V  1  2  2  4  4 W  1  2 X Y 94 89  1 6 Z — Unknown (?) not sequenced  1  2  2  2  2 52 52 52 sum of seq² 9797 97 97 97 97 97 96 95 95 95 95 45 45 45 oomcaa³ 91 96 96 96 88 93 8494 89 95 92 92 11  9 11 mcaa⁴ K A S D T A M Y Y C A R L G G rel. oomcaa⁵94% 99% 99% 99% 91% 96% 87% 98% 94% 100% 97% 97% 24% 20% 24% posoccupied⁶  5  2  2  2  4  2  5  2  2  1  3  4 13 16 14 CDR III aminoacid¹ 98 99 100 A B C D E F G H I J K 101 A  3  4  3  2  1  1  4  2 B C 1  1  1  2  1 D  3  3  1  2  1  1  2  2  1  1  2 37 E  2  1  1  1  1 F 3  3  2  1 26 G 12 12  5  2  4  3 10  2  1  5 H  2  1  1  1 I  2  2  1 1  4  1  1  1  1 K  1  3  1  2 L  1  1  2  5  1  1  1 M  1  1  1  1  1 1 10 N  2  1  1  2  1  2 P  3  1  2  1  1  1  1 Q  1  1  4  2  1  2  3R  2  2  1  2 S  6  4  4  5  3  5  3  2  2  1  1 T  1  2  6  3  3  6  1 1 V  1  1  2  1 W  1  1  2  1  1  1 X Y  3  6  9  8  7  2  1  2  6  8 9  9 10  1 Z —  1  1  2  8 10 16 23 30 30 31 32 30 22  7  2 Unknown (?) 1  1  1  1 not sequenced 52 52 52 52 52 52 52 52 52 52 52 52 52 53 52sum of seq² 45 45 45 45 45 45 45 45 45 45 45 45 45 44 45 oomcaa³ 12 12 9  8 10 16 23 30 30 31 32 30 22 26 37 mcaa⁴ G G Y Y — — — — — — — — — FD rel. oomcaa⁵ 27% 27% 20% 18% 22% 36% 51% 67% 67% 69% 71% 67% 49% 59%82% pos occupied⁶ 18 16 15 16 15 14 11 11  9  8  4  6  6  4  5 FrameworkIV amino acid¹ 102 103 104 105 106 107 108 109 110 111 112 113 sum A  1611 B C 205 D  1 458 E  1 404 F  2 256 G 41 41 1065 H 44 I  9  2 588 K 3 650 L  2 25  1 549 M  8 303 N 64 P  2  1  1 414 Q 34 612 R  3 S  2 4039 1545 T  1 40  8 39 604 V 11 40 41 594 W 43 432 X Y 13 738 Z —  2 635unknown (?)  4 not sequenced 52 54 56 56 56 56 56 56 56 56 56 57 1678sum of seq² 45 43 41 41 41 41 41 41 41 41 41 40 oomcaa³ 13 43 41 34 4140 25 40 39 41 40 39 mcaa⁴ Y W G Q G T L V T V S S rel. oomcaa⁵ 29% 100%100% 83% 100% 98% 61% 98% 95% 100% 98% 98% pos occupied⁶ 10  1  1  4  1 2  3  2  2  1  2  2

TABLE 6G Analysis of V heavy chain subgroup 6 Framework I amino acid¹ 12 3 4 5 6 7 8 9 10 11 12 13 14 15 A B C D E F G 52 67 H I K 68 L 52 68 1 M N P 68 67 Q 52 52 51 52 R  1  1 S 52  1 68 T V 52 66 W X Y Z —unknown (?) not sequenced 22 22 22 22 22 22 22 22  6  6  6  6  6  6  6sum of seq² 52 52 52 52 52 52 52 52 68 68 68 68 68 68 68 oomcaa³ 52 5252 52 51 52 52 52 68 67 68 66 68 67 68 mcaa⁴ Q V Q L Q Q S G P G L V K PS rel. oomcaa⁵ 100% 100% 100% 100% 98% 100% 100% 100% 100% 99% 100% 97%100% 99% 100% pos occupied⁶  1  1  1  1  2  1  1  1  1  2  1  3  1  2  1Framework I amino acid¹ 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 A 1 67 B C 68 D 68 E F  2 G  1 69 H I 64 K L 67  1 68 M N  1 P  1 Q 68 RS 66  1  1 69 69 68 T 68 67 V  1  1  4 70 W  1 X Y Z — unknown (?) notsequenced  6  6  6  6  6  5  5  5  5  5  5  5  5  4  4 sum of seq² 68 6868 68 68 69 69 69 69 69 69 69 69 70 70 oomcaa³ 68 68 67 66 68 67 68 6764 69 69 68 69 70 68 mcaa⁴ Q T L S L T C A I S G D S V S rel. ommcaa⁵100% 100% 99% 97% 100% 97% 99% 97% 93% 100% 100% 99% 100% 100% 97% posoccupied⁶  1  1  2  3  1  3  2  3  3  1  1  2  1  1  2 CDRI Framework IIamino acid¹ 31 A B 32 33 34 35 36 37 38 39 40 41 42 43 A 66 67  1 B C D 1  1 E F  1  1  1 G  3  1  2 H  1 I  2  1 70 K  3  1  1 L  1 M N  2 6670 P 73 Q 72 R  2  1 74 73 S 66 67  3  1 74  1 73 T  2  1  4  1 V  6  2W 74 74 X Y  1  1 Z — unknown (?)  1 not sequenced sum of seq² 74 74 7474 74 74 74 74 74 74 74 74 74 74 74 oomcaa³ 66 66 67 66 67 74 70 74 7074 72 74 73 73 73 mcaa⁴ S N S A A W N W I R Q S P S R rel. ommcaa⁵ 89%89% 91% 89% 91% 100% 95% 100% 95% 100% 97% 100% 99% 99% 99% posoccupied⁶  5  6  3  4  5  1  5  1  4  1  3  1  2  2  2 Framework II CDRII amino acid¹ 44 45 46 47 48 49 50 51 52 A B C 53 54 55 A  1  1 B C D E74 F  2  1  1 G 74 74  1  1 H  1 I K  1 66 L 74 74 M N  1 P Q R 73 72  1 1 S  1 72 T 73  5 V W 74 73 X Y 72 72 Z — 74 unknown (?) not sequencedsum of seq ² 74 74 74 74 74 74 74 74 74 74 74 74 74 74 74 oomcaa³ 74 7474 74 74 74 73 73 72 72 72 74 72 66 73 mcaa⁴ G L E W L G R T Y Y R — S KW rel. ommcaa⁵ 100% 100% 100% 100% 100% 100% 99% 99% 97% 97% 97% 100%97% 89% 99% pos occupied⁶  1  1  1  1  1  1  2  2  2  3  3  1  3  5  2CDR II Framework III amino acid¹ 56 57 58 59 60 61 62 63 64 65 66 67 6869 70 A 73  1  2 B C  1 D 68  1  2 E  1  3  7  1 F  7 G  1  1  8 H  1 I 1 65  2 71 K  1 67  1 L  1  5  2  4 M  1 N  2 65  1  1 69 P  1  1 Q  2 1 R  1  3 73 S  2  2  1  1 73 66  1  2 T  4 69  1 V 58 72  4  2 W X Y60  1 72 Z — unknown (?) not sequenced sum of seq² 74 74 74 74 74 74 7474 74 74 74 74 74 74 74 oomcaa³ 60 65 68 72 73 58 73 72 67 66 73 65 6971 69 mcaa⁴ Y N D Y A V S V K S R I T I N rel. ommcaa⁵ 81% 88% 92% 97%99% 78% 99% 97% 91% 89% 99% 88% 93% 96% 93% pos occupied⁶  7  6  5  3  2 7  2  2  5  2  2  4  4  3  4 Framework III amino acid¹ 71 72 73 74 7576 77 78 79 80 81 82 A B C A  6  1 B C D 73  3 E  2 F 71  1 G H  1  2  1I  1  1 K 70  4 L  1  1 74 72 M  1  1 N 74 63 P 66 Q 72 71 R  1  1  1 S 1 73 74  1 73 T 71  1  2  1 V  1  2  1 73 W X Y Z — unknown (?) notsequenced sum of seq² 74 74 74 74 74 74 74 74 74 74 74 74 74 74 74oomcaa³ 66 73 71 73 70 74 72 71 74 74 71 72 63 73 73 mcaa⁴ P D T S K N QF S L Q L N S V rel. oomcaa⁵ 89% 99% 96% 99% 95% 100% 97% 96% 100% 100%96% 97% 85% 99% 99% pos occupied⁶  4  2  4  2  3  1  3  3  1  1  3  3  7 2  2 Framework III CDR III amino acid¹ 83 84 85 86 87 88 89 90 91 92 9394 95 96 97 A  1 74 69 11  1  3 B C 73  1 D 73 19  4  3 E 73 10  4  2 F 3  1  1  1  1 G  1  1 16  4 15 H  1 I  2  1  2 K  1  1  1  1 L  1  8  4M  2  1 N  1  1  3  1 P 70 10  4 Q  1  1  1 R  1 69  1  7  8 S  1  3  3 5  5  5 T 73 74  1  1  1  4 V 70  3  1  4  5  1 W  1  6  8 X Y 73 70  6 4 Z —  2  3 unknown (?) not sequenced  1  1  2 sum of seq² 74 73 74 7474 74 74 74 74 74 74 74 73 72 71 oomcaa³ 73 70 73 73 74 74 70 73 70 7369 69 19 10 15 mcaa⁴ T P E D T A V Y Y C A R D P G rel. oomcaa⁵ 99% 96%99% 99% 100% 100% 95% 99% 95% 99% 93% 93% 26% 14% 21% pos occupied⁶  2 2  2  2  1  1  3  2  3  2  4  4 14 20 19 CDR III amino acid¹ 98 99 100A B C D E F G H I J K 101 A 12  4  3  2  5  8 10  1 B C  1  1  1  1 D  7 4  3  1  6  1  1  1 62 E  1  2  2  1  2  1 F  1  2  3  2  1 38  4 G 1511  8  6  2  5  1  8  6  1 17 H  1  1  1  1  1  1  1  1 I  2  5  1 K  1 1  1  1 L  2  3  2  1  1  5  8 M  1  5 11 N  2  1  1  1  3  2  1  1  3P  5  3  5  1  1 Q  1  1  1 R  1  8  8  3  1  1  5  1 S  7  6  7  3  4 2  1  1 T  3  4  4  6  3  1  1 V  9  4  9  5  1  1  2 W  3  2  4  4  4X Y  2  2  2  6  6  2  4  2  1  8  8 12 12 Z —  7 14 23 25 33 41 47 5354 57 56 50 28 12  4 unknown (?)  6  1  5 not sequenced  2  1  1  1  1 1  1  1  1  1  1  1  1  1  1 sum of seq² 71 72 72 72 72 72 72 72 72 7272 72 72 72 72 oomcaa³ 15 14 23 25 33 41 47 53 54 57 56 50 28 38 62mcaa⁴ G — — — — — — — — — — — — F D rel. oomcaa⁵ 21% 19% 32% 35% 46% 57%65% 74% 75% 79% 78% 69% 39% 53% 86% pos occupied⁶ 15 17 16 16 13 13 11 8  8  4  5  7  6  6  5 Framework IV amino acid¹ 102 103 104 105 106 107108 109 110 111 112 113 sum A  2 494 B C 147 D  1 403 E 186 F  2  2 150G 49 50 571 H  2 18 I  9  3  1 304 K  1  1 293 L  5 26 632 M  8 31 N 436P  4  6  1 387 Q 40 539 R  2 495 S  4  1  1 43 46 1271 T 45  4 45 640 V21  2 46 48 647 W 65  5 398 X Y 19 518 Z —  2 585 unknown (?) 13 notsequenced  5  8 23 24 23 24 25 25 28 25 28 26 580 sum of seq² 68 65 5049 50 49 48 48 45 48 45 47 oomcaa³ 21 65 49 40 50 45 26 46 45 48 43 46mcaa⁴ V W G Q G T L V T V S S rel. oomcaa⁵ 31% 100% 98% 82% 100% 92% 54%96% 100% 100% 96% 98% pos occupied⁶  9  1  2  4  1  3  7  3  1  1  2  2Appendix to Tables 1A-C

A. REFERENCES OF REARRANGED SEQUENCES REFERENCES OF REARRANGED HUMANKAPPA SEQUENCES USED FOR ALIGNMENT

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1-55. (canceled)
 56. A method of preparing a library of nucleic acids,wherein each nucleic acid encodes an immunoglobulin variable domaincomprising consensus framework sequences, comprising: (a) identifying aplurality of immunoglobulin variable domain amino acid sequences, eachcomprising four consensus framework regions interspaced by threecomplementary determining regions CDR1, CDR2, and CDR3, wherein saidconsensus framework regions have been identified by the following steps:(i) aligning three or more known human immunoglobulin sequences; (ii)identifying the conserved framework regions of said known humanimmunoglobulin sequences; (iii) comparing the amino acids at eachcorresponding position of said conserved framework regions; and (iv)deducing consensus framework regions from said comparing in step(a)(iii); and (b) synthesizing a plurality of nucleic acids encodingsaid plurality of immunoglobulin variable domain amino acid sequencesprovided in step (a), wherein each of said nucleic acids comprises DNAcleavage sites at the boundary between each consensus framework regionand complementary determining region, and wherein each of said cleavagesites is unique within said nucleic acid but common to all nucleic acidsequences of said library at corresponding positions.
 57. The methodaccording to claim 56, wherein said known human immunoglobulin sequencesin step (a)(i) are human Vκ immunoglobulin sequences.
 58. The methodaccording to claim 56, wherein said known human immunoglobulin sequencesin step (a)(i) are human Vλ immunoglobulin sequences.
 59. The methodaccording to claim 56, wherein said known human immunoglobulin sequencesin step (a)(i) are human VH immunoglobulin sequences.
 60. The methodaccording to claim 57, wherein said nucleic acids synthesized in step(b) are selected from the group consisting of Vκ1 (SEQ ID NO:42), Vκ2(SEQ ID NO: 44), Vκ3 (SEQ ID NO: 46), and Vκ4 (SEQ ID NO: 48).
 61. Themethod according to claim 58, wherein said nucleic acids synthesized instep (b) are selected from the group consisting of Vλ1 (SEQ ID NO:50),Vλ2 (SEQ ID NO: 52), and Vλ3 (SEQ ID NO: 54).
 62. The method accordingto claim 59, wherein said nucleic acids synthesized in step (b) areselected from the group consisting of VH1A (SEQ ID NO:56), VH1B (SEQ IDNO: 58), VH2 (SEQ ID NO: 60), VH3 (SEQ ID NO: 62), VH4 (SEQ ID NO: 64),VH5 (SEQ ID NO: 66), and VH6 (SEQ ID NO: 68).
 63. The method accordingto claim 56, further comprising inserting said nucleic acids into anexpression vector.
 64. The method according to claim
 63. The methodaccording to claim 83, wherein said nucleic acids encoding saidimmunoglobulin variable domain amino acid sequences comprise codons thatare frequently used in said host cell.
 65. The method according to claim64, wherein said host cell is E. coli.
 66. The method according to claim65, wherein said expression vector is a phagemid vector.
 67. The methodaccording to claim 56, wherein said CDR1 is selected from the groupconsisting of VH CDR1 germline sequences.
 68. The method according toclaim 67, wherein said CDR1 is selected from the group consisting ofVH1-12-1, VH1-13-16, VH2-31-10, VH3-13-8, VH4-11-7, CH5-12-1, andVH6-35-1.
 69. The method according to claim 56, wherein said CDR1 isselected from the group consisting of Vλ CDR1 germline sequences. 70.The method according to claim 69, wherein said CDR1 is selected from thegroup consisting of VHUMLV86, DPL11, and DPL23.
 71. The method accordingto claim 56, wherein said CDR1 is selected from the group consisting ofVκ CDR1 germline sequences.
 72. The method according to claim 71,wherein said CDR1 is selected from the group consisting of Vκ1-14,Vκ2-6, Vκ33-1, and Vκ4-1.
 73. The method according to claim 56, whereinsaid CDR2 is selected from the group consisting of VH CDR2 germlinesequences.
 74. The method according to claim 73, wherein said CDR2 isselected from the group consisting of VH1-12-1, VH1-13-6, VH2-31-3,VH3-13-8, VH4-11-8, VH4-31-17, VH5-12-1, and VH6-35-1.
 75. The methodaccording to claim 56, wherein said CDR2 is selected from the groupconsisting of Vλ CDR2 germline sequences.
 76. The method according toclaim 75, wherein said CDR2 is selected from the group consisting ofDPL5, DPL12, and HUMLV318.
 77. The method according to claim 56, whereinsaid CDR2 is selected from the group consisting of Vκ CDR2 germlinesequences.
 78. The method according to claim 77, wherein said CDR2 isselected from the group consisting of Vκ1-2, Vκ2-6, Vκ3-4, and Vκ4-1.79. The method according to claim 56, wherein said CDR3 is selected fromrandom amino acid sequences.
 80. The method according to claim 56wherein said CDR3 is an amino acid sequence selected from Vκ CDR3sequences comprising the amino acid sequenceN1-Gln-N-3-N4-N-5-N6-N-7-N8-Thr, wherein: N1 is an amino acid selectedfrom the group consisting of Phe, His, Leu, Met, and Gln; N3 is an aminoacid other than Cys or Pro; N4 is an amino acid selected from the groupconsisting of Asp, Gly, Asn, Ser, and Phe; N5 is an amino acid selectedfrom the group consisting of Asp, Gly, Asn, and Ser; N6 is an amino acidother than Cys; N7 is Pro or Ser; and N8 is an amino acid other thanCys.
 81. The method according to claim 56 wherein said CDR3 is an aminoacid sequence selected from Vλ CDR3 sequences comprising the amino acidsequence Gln-Ser-N-3-Asp-N-5-N6-N-7-N8-N-9-N10, wherein: N1 is an aminoacid selected from the group consisting of Arg, Trp, or Phe; N3 is anamino acid other than Cys or Pro; N5 is an amino acid other then Cys orTrp; N6 is an amino acid other than Cys or Trp; N7 is an amino acidother than Cys or Trp; N8 is an amino acid other than Cys or Trp or N8is absent; N9 is an amino acid other than Cys or Trp or N9 is absent;and N10 is an amino acid other than Cys.